Publications by authors named "Md Shenuarin Bhuiyan"

44 Publications

Disrupted Blood-Brain Barrier and Mitochondrial Impairment by Autotaxin-Lysophosphatidic Acid Axis in Postischemic Stroke.

J Am Heart Assoc 2021 Sep 13:e021511. Epub 2021 Sep 13.

Department of Cellular Biology and Anatomy Louisiana State University Health Sciences Center Shreveport LA.

Background The loss of endothelial integrity increases the risk of intracerebral hemorrhage during ischemic stroke. Adjunct therapeutic targets for reperfusion in ischemic stroke are in need to prevent blood-brain barrier disruption. Recently, we have shown that endothelial permeability is mediated by lysophosphatidic acid (LPA), but the role of autotaxin, which produces LPA, remains unclear in stroke. We investigate whether autotaxin/LPA axis regulates blood-brain barrier integrity after cerebral ischemia. Methods and Results Ischemic stroke was induced in mice by middle cerebral artery occlusion for 90 minutes, followed by 24-hour reperfusion. The therapeutic efficacy of autotaxin/LPA receptor blockade was evaluated using triphenyl tetrazolium chloride staining, Evans blue permeability, infrared imaging, mass spectrometry, and XF24 analyzer to evaluate blood-brain barrier integrity, autotaxin activity, and mitochondrial bioenergetics. In our mouse model of ischemic stroke, the mRNA levels of autotaxin were elevated 1.7-fold following the cerebral ischemia and reperfusion (I/R) group compared with the sham. The enzymatic activity of autotaxin was augmented by 4-fold in the I/R group compared with the sham. Plasma and brain tissues in I/R group showed elevated LPA levels. The I/R group also demonstrated mitochondrial dysfunction, as evidenced by decreased (<0.01) basal oxygen consumption rate, mitochondrial ATP production, and spare respiratory capacity. Treatment with autotaxin inhibitors (HA130 or PF8380) or autotaxin/LPA receptor inhibitor (BrP-LPA) rescued endothelial permeability and mitochondrial dysfunction in I/R group. Conclusions Autotaxin-LPA signaling blockade attenuates blood-brain barrier disruption and mitochondrial function following I/R, suggesting targeting this axis could be a new therapeutic approach toward treating ischemic stroke.
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http://dx.doi.org/10.1161/JAHA.121.021511DOI Listing
September 2021

Sigmar1's Molecular, Cellular, and Biological Functions in Regulating Cellular Pathophysiology.

Front Physiol 2021 7;12:705575. Epub 2021 Jul 7.

Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, United States.

The Sigma 1 receptor (Sigmar1) is a ubiquitously expressed multifunctional inter-organelle signaling chaperone protein playing a diverse role in cellular survival. Recessive mutation in Sigmar1 have been identified as a causative gene for neuronal and neuromuscular disorder. Since the discovery over 40 years ago, Sigmar1 has been shown to contribute to numerous cellular functions, including ion channel regulation, protein quality control, endoplasmic reticulum-mitochondrial communication, lipid metabolism, mitochondrial function, autophagy activation, and involved in cellular survival. Alterations in Sigmar1's subcellular localization, expression, and signaling has been implicated in the progression of a wide range of diseases, such as neurodegenerative diseases, ischemic brain injury, cardiovascular diseases, diabetic retinopathy, cancer, and drug addiction. The goal of this review is to summarize the current knowledge of Sigmar1 biology focusing the recent discoveries on Sigmar1's molecular, cellular, pathophysiological, and biological functions.
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http://dx.doi.org/10.3389/fphys.2021.705575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8293995PMC
July 2021

Editorial: Targeting Cardiac Proteotoxicity.

Front Physiol 2021 25;12:669356. Epub 2021 Mar 25.

Division of Basic Biomedical Sciences, The University of South Dakota Sanford School of Medicine, Vermillion, SD, United States.

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http://dx.doi.org/10.3389/fphys.2021.669356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027103PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Kv1.1 potassium channel subunit deficiency alters ventricular arrhythmia susceptibility, contractility, and repolarization.

Physiol Rep 2021 01;9(1):e14702

Department of Cellular Biology & Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA.

Epilepsy-associated Kv1.1 voltage-gated potassium channel subunits encoded by the Kcna1 gene have traditionally been considered absent in heart, but recent studies reveal they are expressed in cardiomyocytes where they could regulate intrinsic cardiac electrophysiology. Although Kv1.1 now has a demonstrated functional role in atria, its role in the ventricles has never been investigated. In this work, electrophysiological, histological, and gene expression approaches were used to explore the consequences of Kv1.1 deficiency in the ventricles of Kcna1 knockout (KO) mice at the organ, cellular, and molecular levels to determine whether the absence of Kv1.1 leads to ventricular dysfunction that increases the risk of premature or sudden death. When subjected to intracardiac pacing, KO mice showed normal baseline susceptibility to inducible ventricular arrhythmias (VA) but resistance to VA under conditions of sympathetic challenge with isoproterenol. Echocardiography revealed cardiac contractile dysfunction manifesting as decreased ejection fraction and fractional shortening. In whole-cell patch-clamp recordings, KO ventricular cardiomyocytes exhibited action potential prolongation indicative of impaired repolarization. Imaging, histological, and transcript analyses showed no evidence of structural or channel gene expression remodeling, suggesting that the observed deficits are likely electrogenic due to Kv1.1 deficiency. Immunoblots of patient heart samples detected the presence of Kv1.1 at relatively high levels, implying that Kv1.1 contributes to human cardiac electrophysiology. Taken together, this work describes an important functional role for Kv1.1 in ventricles where its absence causes repolarization and contractility deficits but reduced susceptibility to arrhythmia under conditions of sympathetic drive.
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http://dx.doi.org/10.14814/phy2.14702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798052PMC
January 2021

Dysfunctional Mitochondrial Dynamic and Oxidative Phosphorylation Precedes Cardiac Dysfunction in R120G-αB-Crystallin-Induced Desmin-Related Cardiomyopathy.

J Am Heart Assoc 2020 12 19;9(23):e017195. Epub 2020 Nov 19.

Department of Pathology and Translational Pathobiology Louisiana State University Health Sciences Center Shreveport LA.

Background The mutated α-B-Crystallin (CryAB) mouse model of desmin-related myopathy (DRM) shows an age-dependent onset of pathologic cardiac remodeling and progression of heart failure. CryAB expression in cardiomyocytes affects the mitochondrial spatial organization within the myofibrils, but the molecular perturbation within the mitochondria in the relation of the overall course of the proteotoxic disease remains unclear. Methods and Results CryAB mice show an accumulation of electron-dense aggregates and myofibrillar degeneration associated with the development of cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause cardiac contractility impairment, the molecular mechanism of cardiomyocyte death remains elusive. Here, we explore early pathological processes within the mitochondria contributing to the contractile dysfunction and determine the pathogenic basis for the heart failure observed in the CryAB mice. In the present study, we report that the CryAB mice transgenic hearts undergo altered mitochondrial dynamics associated with increased level of dynamin-related protein 1 and decreased level of optic atrophy type 1 as well as mitofusin 1 over the disease process. In association with these changes, an altered level of the components of mitochondrial oxidative phosphorylation and pyruvate dehydrogenase complex regulatory proteins occurs before the manifestation of pathologic adverse remodeling in the CryAB hearts. Mitochondria isolated from CryAB transgenic hearts without visible pathology show decreased electron transport chain complex activities and mitochondrial respiration. Taken together, we demonstrated the involvement of mitochondria in the pathologic remodeling and progression of DRM-associated cellular dysfunction. Conclusions Mitochondrial dysfunction in the form of altered mitochondrial dynamics, oxidative phosphorylation and pyruvate dehydrogenase complex proteins level, abnormal electron transport chain complex activities, and mitochondrial respiration are evident on the CryAB hearts before the onset of detectable pathologies and development of cardiac contractile dysfunction.
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http://dx.doi.org/10.1161/JAHA.120.017195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763772PMC
December 2020

Methamphetamine induces cardiomyopathy by Sigmar1 inhibition-dependent impairment of mitochondrial dynamics and function.

Commun Biol 2020 11 17;3(1):682. Epub 2020 Nov 17.

Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71103, USA.

Methamphetamine-associated cardiomyopathy is the leading cause of death linked with illicit drug use. Here we show that Sigmar1 is a therapeutic target for methamphetamine-associated cardiomyopathy and defined the molecular mechanisms using autopsy samples of human hearts, and a mouse model of "binge and crash" methamphetamine administration. Sigmar1 expression is significantly decreased in the hearts of human methamphetamine users and those of "binge and crash" methamphetamine-treated mice. The hearts of methamphetamine users also show signs of cardiomyopathy, including cellular injury, fibrosis, and enlargement of the heart. In addition, mice expose to "binge and crash" methamphetamine develop cardiac hypertrophy, fibrotic remodeling, and mitochondrial dysfunction leading to contractile dysfunction. Methamphetamine treatment inhibits Sigmar1, resulting in inactivation of the cAMP response element-binding protein (CREB), decreased expression of mitochondrial fission 1 protein (FIS1), and ultimately alteration of mitochondrial dynamics and function. Therefore, Sigmar1 is a viable therapeutic agent for protection against methamphetamine-associated cardiomyopathy.
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http://dx.doi.org/10.1038/s42003-020-01408-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7673131PMC
November 2020

Chemical Architecture of Block Copolymers Differentially Abrogate Cardiotoxicity and Maintain the Anticancer Efficacy of Doxorubicin.

Mol Pharm 2020 12 5;17(12):4676-4690. Epub 2020 Nov 5.

Department of Coatings and Polymeric Materials, North Dakota State University, Fargo, North Dakota 58108, United States.

The molecular architecture of pH-responsive amphiphilic block copolymers, their self-assembly behavior to form nanoparticles (NPs), and doxorubicin (DOX)-loading technique govern the extent of DOX-induced cardiotoxicity. We observed that the choice of pH-sensitive tertiary amines, surface charge, and DOX-loading techniques within the self-assembled NPs strongly influence the release and stimulation of DOX-induced cardiotoxicity in primary cardiomyocytes. However, covalent conjugation of DOX to a pH-sensitive nanocarrier through a "conditionally unstable amide" linkage (PCPY-cDOX; PC = polycarbonate and PY = 2-pyrrolidine-1-yl-ethyl-amine) significantly reduced the cardiotoxicity of DOX in cardiomyocytes as compared to noncovalently encapsulated DOX NPs (PCPY-eDOX). When these formulations were tested for drug release in serum-containing media, the PCPY-cDOX systems showed prolonged control over drug release (for ∼72 h) at acidic pH compared to DOX-encapsulated nanocarriers, as expected. We found that DOX-encapsulated nanoformulations triggered cardiotoxicity in primary cardiomyocytes more acutely, while conjugated systems such as PCPY-cDOX prevented cardiotoxicity by disabling the nuclear entry of the drug. Using 2D and 3D (spheroid) cultures of an ER + breast cancer cell line (MCF-7) and a triple-negative breast cancer cell line (MDA-MB-231), we unravel that, similar to encapsulated systems (PCPY-eDOX-type) as reported earlier, the PCPY-cDOX system suppresses cellular proliferation in both cell lines and enhances trafficking through 3D spheroids of MDA-MB-231 cells. Collectively, our studies indicate that PCPY-cDOX is less cardiotoxic as compared to noncovalently encapsulated variants without compromising the chemotherapeutic properties of the drug. Thus, our studies suggest that the appropriate selection of the nanocarrier for DOX delivery may prove fruitful in shifting the balance between low cardiotoxicity and triggering the chemotherapeutic potency of DOX.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00963DOI Listing
December 2020

SOD2 deficiency in cardiomyocytes defines defective mitochondrial bioenergetics as a cause of lethal dilated cardiomyopathy.

Redox Biol 2020 10 30;37:101740. Epub 2020 Sep 30.

Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA. Electronic address:

Electrophilic aldehyde (4-hydroxynonenal; 4-HNE), formed after lipid peroxidation, is a mediator of mitochondrial dysfunction and implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese superoxide dismutase (MnSOD), a nuclear-encoded antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O) in mitochondria. To study the role of MnSOD in the myocardium, we generated a cardiomyocyte-specific SOD2 (SOD2Δ) deficient mouse strain. Unlike global SOD2 knockout mice, SOD2Δ mice reached adolescence; however, they die at ~4 months of age due to heart failure. Ultrastructural analysis of SOD2Δ hearts revealed altered mitochondrial architecture, with prominent disruption of the cristae and vacuole formation. Noninvasive echocardiographic measurements in SOD2 mice showed dilated cardiomyopathic features such as decreased ejection fraction and fractional shortening along with increased left ventricular internal diameter. An increased incidence of ventricular tachycardia was observed during electrophysiological studies of the heart in SOD2Δ mice. Oxidative phosphorylation (OXPHOS) measurement using a Seahorse XF analyzer in SOD2Δ neonatal cardiomyocytes and adult cardiac mitochondria displayed reduced O consumption, particularly during basal conditions and after the addition of FCCP (H ionophore/uncoupler), compared to that in SOD2fl hearts. Measurement of extracellular acidification (ECAR) to examine glycolysis in these cells showed a pattern precisely opposite that of the oxygen consumption rate (OCR) among SOD2Δ mice compared to their SOD2 littermates. Analysis of the activity of the electron transport chain complex identified a reduction in Complex I and Complex V activity in SOD2Δ compared to SOD2fl mice. We demonstrated that a deficiency of SOD2 increases reactive oxygen species (ROS), leading to subsequent overproduction of 4-HNE inside mitochondria. Mechanistically, proteins in the mitochondrial respiratory chain complex and TCA cycle (NDUFS2, SDHA, ATP5B, and DLD) were the target of 4-HNE adduction in SOD2Δ hearts. Our findings suggest that the SOD2 mediated 4-HNE signaling nexus may play an important role in cardiomyopathy.
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http://dx.doi.org/10.1016/j.redox.2020.101740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559509PMC
October 2020

Molecular Perspectives of Mitochondrial Adaptations and Their Role in Cardiac Proteostasis.

Front Physiol 2020 27;11:1054. Epub 2020 Aug 27.

Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center, Shreveport, LA, United States.

Mitochondria are the key to properly functioning energy generation in the metabolically demanding cardiomyocytes and thus essential to healthy heart contractility on a beat-to-beat basis. Mitochondria being the central organelle for cellular metabolism and signaling in the heart, its dysfunction leads to cardiovascular disease. The healthy mitochondrial functioning critical to maintaining cardiomyocyte viability and contractility is accomplished by adaptive changes in the dynamics, biogenesis, and degradation of the mitochondria to ensure cellular proteostasis. Recent compelling evidence suggests that the classical protein quality control system in cardiomyocytes is also under constant mitochondrial control, either directly or indirectly. Impairment of cytosolic protein quality control may affect the position of the mitochondria in relation to other organelles, as well as mitochondrial morphology and function, and could also activate mitochondrial proteostasis. Despite a growing interest in the mitochondrial quality control system, very little information is available about the molecular function of mitochondria in cardiac proteostasis. In this review, we bring together current understanding of the adaptations and role of the mitochondria in cardiac proteostasis and describe the adaptive/maladaptive changes observed in the mitochondrial network required to maintain proteomic integrity. We also highlight the key mitochondrial signaling pathways activated in response to proteotoxic stress as a cellular mechanism to protect the heart from proteotoxicity. A deeper understanding of the molecular mechanisms of mitochondrial adaptations and their role in cardiac proteostasis will help to develop future therapeutics to protect the heart from cardiovascular diseases.
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http://dx.doi.org/10.3389/fphys.2020.01054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481364PMC
August 2020

Pleiotropic effects of mdivi-1 in altering mitochondrial dynamics, respiration, and autophagy in cardiomyocytes.

Redox Biol 2020 09 26;36:101660. Epub 2020 Jul 26.

Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, 71103, USA; Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center, Shreveport, LA, 71103, USA. Electronic address:

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion events to adapt to changes in the cellular environment. Aberrant mitochondrial fission has been associated with several types of cardiovascular dysfunction; inhibition of pathologically aberrant mitochondrial fission has been shown to be cardioprotective. Pathological fission is mediated by the excessive activation of GTPase dynamin-related protein 1 (Drp1), making it an attractive therapeutic target in numerous cardiovascular diseases. Mitochondrial division inhibitor (mdivi-1) is widely used small molecule reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate injury. The purpose of our study was to understand the pleiotropic effects of mdivi-1 on mitochondrial dynamics, mitochondrial respiration, electron transport activities, and macro-autophagy. In this study, we found that mdivi-1 treatment decreased Drp1 expression, proteolytically cleaved L-OPA1, and altered the expression of OXPHOS complex proteins, resulting in increased superoxide production. The altered expression of OXPHOS complex proteins may be directly associated with decreased Drp1 expression, as Drp1 siRNA knockdown in cardiomyocytes showed similar effects. Results from an autophagy flux assay showed that mdivi-1 induced impaired autophagy flux that could be restored by Atg7 overexpression, suggesting that mdivi-1 mediated inhibition of macro-autophagy in cardiomyocytes. Treatment with mdivi-1 resulted in increased expression of p62, which is required for Atg7 overexpression-induced rescue of mdivi-1-mediated impaired autophagy flux. In addition, mdivi-1-dependent proteolytic processing of L-OPA1 was associated with increased mitochondrial superoxide production and altered expression of mitochondrial serine/proteases. Overall, the novel pleiotropic effect of mdivi-1 in cardiomyocytes included proteolytically cleaved L-OPA1, altered expression of OXPHOS complex proteins, and increased superoxide production, which together resulted in defects in mitochondrial respiration and inhibition of macro-autophagy.
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http://dx.doi.org/10.1016/j.redox.2020.101660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396909PMC
September 2020

Neurogranin regulates eNOS function and endothelial activation.

Redox Biol 2020 07 5;34:101487. Epub 2020 Mar 5.

Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA, 71130, USA. Electronic address:

Endothelial nitric oxide (NO) is a critical mediator of vascular function and vascular remodeling. NO is produced by endothelial nitric oxide synthase (eNOS), which is activated by calcium (Ca)-dependent and Ca-independent pathways. Here, we report that neurogranin (Ng), which regulates Ca-calmodulin (CaM) signaling in the brain, is uniquely expressed in endothelial cells (EC) of human and mouse vasculature, and is also required for eNOS regulation. To test the role of Ng in eNOS activation, Ng knockdown in human aortic endothelial cells (HAEC) was performed using Ng SiRNA along with Ng knockout (Ng ) in mice. Depletion of Ng expression decreased eNOS activity in HAEC and NO production in mice. We show that Ng expression was decreased by short-term laminar flow and long-them oscillating flow shear stress, and that Ng siRNA with shear stress decreased eNOS expression as well as eNOS phosphorylation at S1177. We further reveled that lack of Ng expression decreases both AKT-dependent eNOS phosphorylation, NF-κB-mediated eNOS expression, and promotes endothelial activation. Our findings also indicate that Ng modulates Ca-dependent calcineurin (CaN) activity, which suppresses Ca-independent AKT-dependent eNOS signaling. Moreover, deletion of Ng in mice also reduced eNOS activity and caused endothelial dysfunction in flow-mediated dilation experiments. Our results demonstrate that Ng plays a crucial role in Ca-CaM-dependent eNOS regulation and contributes to vascular remodeling, which is important for the pathophysiology of cardiovascular disease.
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http://dx.doi.org/10.1016/j.redox.2020.101487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327963PMC
July 2020

Methamphetamine Use and Cardiovascular Disease.

Arterioscler Thromb Vasc Biol 2019 09 21;39(9):1739-1746. Epub 2019 Aug 21.

From the Departments of Pathology and Translational Pathobiology (C.G.K., M.S.B., G.K.K., J.G.T., A.W.O.), LSU Health Sciences Center, Shreveport, LA.

While the opioid epidemic has garnered significant attention, the use of methamphetamines is growing worldwide independent of wealth or region. Following overdose and accidents, the leading cause of death in methamphetamine users is cardiovascular disease, because of significant effects of methamphetamine on vasoconstriction, pulmonary hypertension, atherosclerotic plaque formation, cardiac arrhythmias, and cardiomyopathy. In this review, we examine the current literature on methamphetamine-induced changes in cardiovascular health, discuss the potential mechanisms regulating these varied effects, and highlight our deficiencies in understanding how to treat methamphetamine-associated cardiovascular dysfunction.
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http://dx.doi.org/10.1161/ATVBAHA.119.312461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709697PMC
September 2019

Doxorubicin-induced cardiomyopathy associated with inhibition of autophagic degradation process and defects in mitochondrial respiration.

Sci Rep 2019 02 14;9(1):2002. Epub 2019 Feb 14.

Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71103, USA.

Doxorubicin (Dox) is a highly effective anticancer drug but cause acute ventricular dysfunction, and also induce late-onset cardiomyopathy and heart failure. Despite extensive studies, the pathogenic sequelae leading to the progression of Dox-associated cardiomyopathy remains unknown. We assessed temporal changes in autophagy, mitochondrial dynamics, and bioenergetics in mouse models of acute and chronic Dox-cardiomyopathy. Time course study of acute Dox-treatment showed accumulation of LC3B II in heart lysates. Autophagy flux assays confirmed that the Dox-induced accumulation of autophagosomes occurs due to blockage of the lysosomal degradation process. Dox-induced autophagosomes and autolysosome accumulation were confirmed in vivo by using GFP-LC3 and mRFP-GFP-LC3 transgenic (Tg) mice. Mitochondria isolated from acute Dox-treated hearts showed significant suppression of oxygen consumption rate (OCR). Chronic Dox-cardiotoxicity also exhibited time-dependent accumulation of LC3B II levels and increased accumulation of green puncta in GFP-LC3 Tg hearts. Mitochondria isolated from chronic Dox-treated hearts also showed significant suppression of mitochondrial OCR. The in vivo impairment of autophagic degradation process and mitochondrial dysfunction data were confirmed in vitro using cultured neonatal cardiomyocytes. Both acute and chronic Dox-associated cardiomyopathy involves a multifocal disease process resulting from autophagosomes and autolysosomes accumulation, altered expression of mitochondrial dynamics and oxidative phosphorylation regulatory proteins, and mitochondrial respiratory dysfunction.
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http://dx.doi.org/10.1038/s41598-018-37862-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376057PMC
February 2019

Myofibroblast-Specific TGFβ Receptor II Signaling in the Fibrotic Response to Cardiac Myosin Binding Protein C-Induced Cardiomyopathy.

Circ Res 2018 12;123(12):1285-1297

From the Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital, OH (Q.M., B.B., H.O., I.V.-A., K.S.-W., J.G., J.D.M., B.C.B., J.R.).

Rationale: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc3). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy-one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease.

Objective: TGFβ (transforming growth factor-β) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFβ signaling during chronic Mybpc3 expression.

Methods And Results: To specifically block TGFβ signaling only in the activated myofibroblasts in Mybpc3 transgenic mice and quadruple compound mutant mice were generated, in which the TGFβ receptor II (TβRII) alleles ( Tgfbr2) were ablated using the periostin ( Postn) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc3 transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc3 mouse survival but failed to reverse cardiac hypertrophy.

Conclusions: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc3 were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFβ signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309316PMC
December 2018

Cardiac Dysfunction in the Sigma 1 Receptor Knockout Mouse Associated With Impaired Mitochondrial Dynamics and Bioenergetics.

J Am Heart Assoc 2018 10;7(20):e009775

1 Department of Pathology and Translational Pathobiology Louisiana State University Health Sciences Center Shreveport LA.

Background The Sigma 1 receptor (Sigmar1) functions as an interorganelle signaling molecule and elicits cytoprotective functions. The presence of Sigmar1 in the heart was first reported on the basis of a ligand-binding assay, and all studies to date have been limited to pharmacological approaches using less-selective ligands for Sigmar1. However, the physiological function of cardiac Sigmar1 remains unknown. We investigated the physiological function of Sigmar1 in regulating cardiac hemodynamics using the Sigmar1 knockout mouse (Sigmar1). Methods and Results Sigmar1 hearts at 3 to 4 months of age showed significantly increased contractility as assessed by left ventricular catheterization with stimulation by increasing doses of a β-adrenoceptor agonist. Noninvasive echocardiographic measurements were also used to measure cardiac function over time, and the data showed the development of cardiac contractile dysfunction in Sigmar1 hearts as the animals aged. Histochemistry demonstrated significant cardiac fibrosis, collagen deposition, and increased periostin in the Sigmar1 hearts compared with wild-type hearts. Ultrastructural analysis of Sigmar1 cardiomyocytes revealed an irregularly shaped, highly fused mitochondrial network with abnormal cristae. Mitochondrial size was larger in Sigmar1 hearts, resulting in decreased numbers of mitochondria per microscopic field. In addition, Sigmar1 hearts showed altered expression of mitochondrial dynamics regulatory proteins. Real-time oxygen consumption rates in isolated mitochondria showed reduced respiratory function in Sigmar1 hearts compared with wild-type hearts. Conclusions We demonstrate a potential function of Sigmar1 in regulating normal mitochondrial organization and size in the heart. Sigmar1 loss of function led to mitochondrial dysfunction, abnormal mitochondrial architecture, and adverse cardiac remodeling, culminating in cardiac contractile dysfunction.
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http://dx.doi.org/10.1161/JAHA.118.009775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474981PMC
October 2018

Cardiac Fibrosis in Proteotoxic Cardiac Disease is Dependent Upon Myofibroblast TGF -β Signaling.

J Am Heart Assoc 2018 10;7(20):e010013

1 Division of Molecular Cardiovascular Biology Cincinnati Children's Hospital Cincinnati OH.

Background Transforming growth factor beta ( TGF -β) is an important cytokine in mediating the cardiac fibrosis that often accompanies pathogenic cardiac remodeling. Cardiomyocyte-specific expression of a mutant αB-crystallin (Cry AB ), which causes human desmin-related cardiomyopathy, results in significant cardiac fibrosis. During onset of fibrosis, fibroblasts are activated to the so-called myofibroblast state and TGF -β binding mediates an essential signaling pathway underlying this process. Here, we test the hypothesis that fibroblast-based TGF -β signaling can result in significant cardiac fibrosis in a disease model of cardiac proteotoxicity that has an exclusive cardiomyocyte-based etiology. Methods and Results Against the background of cardiomyocyte-restricted expression of Cry AB , we have partially ablated TGF -β signaling in cardiac myofibroblasts to observe whether cardiac fibrosis is reduced despite the ongoing pathogenic stimulus of Cry AB production. Transgenic Cry AB mice were crossed with mice containing a floxed allele of TGF -β receptor 2 ( Tgfbr2 ). The double transgenic animals were subsequently crossed to another transgenic line in which Cre expression was driven from the periostin locus ( Postn) so that Tgfbr2 would be ablated with myofibroblast conversion. Structural and functional assays were then used to determine whether general fibrosis was affected and cardiac function rescued in Cry AB mice lacking Tgfbr2 in the myofibroblasts. Ablation of myofibroblast specific TGF -β signaling led to decreased morbidity in a proteotoxic disease resulting from cardiomyocyte autonomous expression of Cry AB . Cardiac fibrosis was decreased and hypertrophy was also significantly attenuated, with a significant improvement in survival probability over time, even though the primary proteotoxic insult continued. Conclusions Myofibroblast-targeted knockdown of Tgfbr2 signaling resulted in reduced fibrosis and improved cardiac function, leading to improved probability of survival.
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http://dx.doi.org/10.1161/JAHA.118.010013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474972PMC
October 2018

Aberrant Mitochondrial Fission Is Maladaptive in Desmin Mutation-Induced Cardiac Proteotoxicity.

J Am Heart Assoc 2018 07 9;7(14). Epub 2018 Jul 9.

Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center, Shreveport, LA

Background: Desmin filament proteins interlink the contractile myofibrillar apparatus with mitochondria, nuclei and the sarcolemma. Mutations in the human desmin gene cause cardiac disease, remodeling, and heart failure but the pathophysiological mechanisms remain unknown.

Methods And Results: Cardiomyocyte-specific overexpression of mutated desmin (a 7 amino acid deletion R172-E178, D7-Des Tg) causes accumulations of electron-dense aggregates and myofibrillar degeneration associated with cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause impairment of cardiac contractility, the molecular mechanism of cardiomyocyte death remains elusive. In the present study, we report that the D7-Des Tg mouse hearts undergo aberrant mitochondrial fission associated with increased expression of mitochondrial fission regulatory proteins. Mitochondria isolated from D7-Des Tg hearts showed decreased mitochondrial respiration and increased apoptotic cell death. Overexpression of mutant desmin by adenoviral infection in cultured cardiomyocytes led to increased mitochondrial fission, inhibition of mitochondrial respiration, and activation of cellular toxicity. Inhibition of mitochondrial fission by mitochondrial division inhibitor mdivi-1 significantly improved mitochondrial respiration and inhibited cellular toxicity associated with D7-Des overexpression in cardiomyocytes.

Conclusions: Aberrant mitochondrial fission results in mitochondrial respiratory defects and apoptotic cell death in D7-Des Tg hearts. Inhibition of aberrant mitochondrial fission using mitochondrial division inhibitor significantly preserved mitochondrial function and decreased apoptotic cell death. Taken together, our study shows that maladaptive aberrant mitochondrial fission causes desminopathy-associated cellular dysfunction.
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http://dx.doi.org/10.1161/JAHA.118.009289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6064863PMC
July 2018

Cardiac-specific inactivation of LPP3 in mice leads to myocardial dysfunction and heart failure.

Redox Biol 2018 04 28;14:261-271. Epub 2017 Sep 28.

Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, USA. Electronic address:

Lipid Phosphate phosphatase 3 (LPP3), encoded by the Plpp3 gene, is an enzyme that dephosphorylates the bioactive lipid mediator lysophosphatidic acid (LPA). To study the role of LPP3 in the myocardium, we generated a cardiac specific Plpp3 deficient mouse strain. Although these mice were viable at birth in contrast to global Plpp3 knockout mice, they showed increased mortality ~ 8 months. LPP3 deficient mice had enlarged hearts with reduced left ventricular performance as seen by echocardiography. Cardiac specific Plpp3 deficient mice had longer ventricular effective refractory periods compared to their Plpp3 littermates. We observed that lack of Lpp3 enhanced cardiomyocyte hypertrophy based on analysis of cell surface area. We found that lack of Lpp3 signaling was mediated through the activation of Rho and phospho-ERK pathways. There are increased levels of fetal genes Natriuretic Peptide A and B (Nppa and Nppb) expression indicating myocardial dysfunction. These mice also demonstrate mitochondrial dysfunction as evidenced by a significant decrease (P < 0.001) in the basal oxygen consumption rate, mitochondrial ATP production, and spare respiratory capacity as measured through mitochondrial bioenergetics. Histology and transmission electron microscopy of these hearts showed disrupted sarcomere organization and intercalated disc, with a prominent disruption of the cristae and vacuole formation in the mitochondria. Our findings suggest that LPA/LPP3-signaling nexus plays an important role in normal function of cardiomyocytes.
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http://dx.doi.org/10.1016/j.redox.2017.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635346PMC
April 2018

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes.

Biosci Rep 2017 Aug 16;37(4). Epub 2017 Jul 16.

Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center, Shreveport, LA 71103, U.S.A.

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes.
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http://dx.doi.org/10.1042/BSR20170898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518542PMC
August 2017

Corticosteroids Mediate Heart Failure-Induced Depression through Reduced σ1-Receptor Expression.

PLoS One 2016 14;11(10):e0163992. Epub 2016 Oct 14.

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai, Japan.

Cardiovascular diseases are risk factors for depression in humans. We recently proposed that σ1 receptor (σ1R) stimulation rescued cardiac hypertrophy and heart failure induced by transverse aortic constriction (TAC) in mice. Importantly, σ1R stimulation reportedly ameliorates depression-like behaviors in rodents. Thus, we hypothesized that impaired σ1R activity in brain triggers depression-like behaviors in animals with cardiovascular disease. Indeed, here we found that cardiac hypertrophy and heart failure induced by TAC were associated with depression-like behaviors concomitant with downregulation of σ1R expression in brain 6 weeks after surgery. σ1R levels significantly decreased in astrocytes in both the hippocampal CA1 region and dentate gyrus. Oral administration of the specific σ1R agonist SA4503 (0.3-1.0mg/kg) significantly improved TAC-induced depression-like behaviors concomitant with rescued astrocytic σ1R expression in CA1 and the dentate gyrus. Plasma corticosterone levels significantly increased 6 weeks after TAC, and chronic treatment of mice with corticosterone for 3 weeks elicited depression-like behaviors concomitant with reduced astrocytic σ1R expression in hippocampus. Furthermore, the glucocorticoid receptor antagonist mifepristone antagonized depressive-like behaviors and ameliorated decreased hippocampal σ1R expression in TAC mice. We conclude that elevated corticosterone levels trigger hippocampal σ1R downregulation and that σ1R stimulation with SA4503 is an attractive therapy to improve not only cardiac dysfunction but depression-like behaviors associated with heart failure.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163992PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065174PMC
June 2017

In vivo definition of cardiac myosin-binding protein C's critical interactions with myosin.

Pflugers Arch 2016 10 27;468(10):1685-95. Epub 2016 Aug 27.

Department of Pediatrics, Division of Molecular Cardiovascular Biology, The Heart Institute, Cincinnati Children's Hospital Medical Center, MLC 7020, 240 Albert Sabin Way, Cincinnati, OH, 45229, USA.

Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.
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http://dx.doi.org/10.1007/s00424-016-1873-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308206PMC
October 2016

Haloperidol aggravates transverse aortic constriction-induced heart failure via mitochondrial dysfunction.

J Pharmacol Sci 2016 Jul 4;131(3):172-83. Epub 2016 Jun 4.

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai, Japan. Electronic address:

Haloperidol is an antipsychotic drug that inhibits the dopamine D2 receptor among others. Haloperidol also binds the sigma-1 receptor (σ1R) and inhibits it irreversibly. A serious outcome of haloperidol treatment of schizophrenia patients is death due to sudden cardiac failure. Although the cause remains unclear, we hypothesized that these effects were mediated by chronic haloperidol inhibition of cardiac σ1R. To test this, we treated neonatal rat cardiomyocytes with haloperidol, exposed them to angiotensin II and assessed hypertrophy, σ1R expression, mitochondrial Ca(2+) transport and ATP levels. In this context, haloperidol treatment altered mitochondrial Ca(2+) transport resulting in decreased ATP content by inactivating cardiac σ1R and/or reducing its expression. We also performed transverse aortic constriction (TAC) and then treated mice with haloperidol. After two weeks, haloperidol-treated mice showed enhanced heart failure marked by deteriorated cardiac function, reduced ATP production and increasing mortality relative to TAC only mice. ATP supplementation via sodium pyruvate rescued phenotypes seen in haloperidol-treated TAC mice. We conclude that σ1R inactivation or downregulation in response to haloperidol treatment impairs mitochondrial Ca(2+) mobilization, depleting ATP depletion from cardiomyocytes. These findings suggest a novel approach to mitigate haloperidol-related adverse effects in schizophrenia patients by ATP supplementation.
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http://dx.doi.org/10.1016/j.jphs.2016.05.012DOI Listing
July 2016

Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy.

Proc Natl Acad Sci U S A 2014 Dec 17;111(48):E5178-86. Epub 2014 Nov 17.

The Heart Institute, Department of Pediatrics, Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229; and

Proteinopathy causes cardiac disease, remodeling, and heart failure but the pathological mechanisms remain obscure. Mutated αB-crystallin (CryAB(R120G)), when expressed only in cardiomyocytes in transgenic (TG) mice, causes desmin-related cardiomyopathy, a protein conformational disorder. The disease is characterized by the accumulation of toxic misfolded protein species that present as perinuclear aggregates known as aggresomes. Previously, we have used the CryAB(R120G) model to determine the underlying processes that result in these pathologic accumulations and to explore potential therapeutic windows that might be used to decrease proteotoxicity. We noted that total ventricular protein is hypoacetylated while hyperacetylation of α-tubulin, a substrate of histone deacetylase 6 (HDAC6) occurs. HDAC6 has critical roles in protein trafficking and autophagy, but its function in the heart is obscure. Here, we test the hypothesis that tubulin acetylation is an adaptive process in cardiomyocytes. By modulating HDAC6 levels and/or activity genetically and pharmacologically, we determined the effects of tubulin acetylation on aggregate formation in CryAB(R120G) cardiomyocytes. Increasing HDAC6 accelerated aggregate formation, whereas siRNA-mediated knockdown or pharmacological inhibition ameliorated the process. HDAC inhibition in vivo induced tubulin hyperacetylation in CryAB(R120G) TG hearts, which prevented aggregate formation and significantly improved cardiac function. HDAC6 inhibition also increased autophagic flux in cardiomyocytes, and increased autophagy in the diseased heart correlated with increased tubulin acetylation, suggesting that autophagy induction might underlie the observed cardioprotection. Taken together, our data suggest a mechanistic link between tubulin hyperacetylation and autophagy induction and points to HDAC6 as a viable therapeutic target in cardiovascular disease.
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http://dx.doi.org/10.1073/pnas.1415589111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260547PMC
December 2014

Fluvoxamine rescues mitochondrial Ca2+ transport and ATP production through σ(1)-receptor in hypertrophic cardiomyocytes.

Life Sci 2014 Jan 25;95(2):89-100. Epub 2013 Dec 25.

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan. Electronic address:

Aims: We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ1-receptor (σ1R), ameliorates cardiac hypertrophy and dysfunction via σ1R stimulation. Although σ1R on non-cardiomyocytes interacts with the IP3 receptor (IP3R) to promote mitochondrial Ca(2+) transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main Methods: Here we performed Ca(2+) imaging and measured ATP production to define the role of σ1Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca(2+) transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key Finding: These cardiomyocytes exhibited imbalances in expression levels of σ1R and IP3R and impairments in both phenylephrine-induced mitochondrial Ca(2+) mobilization from the SR and ATP production. Interestingly, σ1R stimulation with fluvoxamine rescued impaired mitochondrial Ca(2+) mobilization and ATP production, an effect abolished by treatment of cells with the σ1R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ1Rs suppressed intracellular Ca(2+) mobilization through IP3Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance: These results suggest that σ1R stimulation with fluvoxamine promotes SR-mitochondrial Ca(2+) transport and mitochondrial ATP production, whereas σ1R stimulation suppresses intracellular Ca(2+) overload through IP3Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ1R agonists including fluvoxamine.
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http://dx.doi.org/10.1016/j.lfs.2013.12.019DOI Listing
January 2014

Enhanced autophagy ameliorates cardiac proteinopathy.

J Clin Invest 2013 Dec 1;123(12):5284-97. Epub 2013 Nov 1.

Basal autophagy is a crucial mechanism in cellular homeostasis, underlying both normal cellular recycling and the clearance of damaged or misfolded proteins, organelles and aggregates. We showed here that enhanced levels of autophagy induced by either autophagic gene overexpression or voluntary exercise ameliorated desmin-related cardiomyopathy (DRC). To increase levels of basal autophagy, we generated an inducible Tg mouse expressing autophagy-related 7 (Atg7), a critical and rate-limiting autophagy protein. Hearts from these mice had enhanced autophagy, but normal morphology and function. We crossed these mice with CryABR120G mice, a model of DRC in which autophagy is significantly attenuated in the heart, to test the functional significance of autophagy activation in a proteotoxic model of heart failure. Sustained Atg7-induced autophagy in the CryABR120G hearts decreased interstitial fibrosis, ameliorated ventricular dysfunction, decreased cardiac hypertrophy, reduced intracellular aggregates and prolonged survival. To determine whether different methods of autophagy upregulation have additive or even synergistic benefits, we subjected the autophagy-deficient CryABR120G mice and the Atg7-crossed CryABR120G mice to voluntary exercise, which also upregulates autophagy. The entire exercised Atg7-crossed CryABR120G cohort survived to 7 months. These findings suggest that activating autophagy may be a viable therapeutic strategy for improving cardiac performance under proteotoxic conditions.
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http://dx.doi.org/10.1172/JCI70877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859422PMC
December 2013

Diverse regulation of IP3 and ryanodine receptors by pentazocine through σ1-receptor in cardiomyocytes.

Am J Physiol Heart Circ Physiol 2013 Oct 9;305(8):H1201-12. Epub 2013 Aug 9.

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan;

Although pentazocine binds to σ1-receptor (σ1R) with high affinity, the physiological relevance of its binding remains unclear. We first confirmed that σ1R stimulation with pentazocine rescues contractile dysfunction following pressure overload (PO)-induced cardiac hypertrophy ovariectomized (OVX) female rats. In in vivo studies, vehicle, pentazocine (0.5-1.0 mg/kg ip), and NE-100 (1.0 mg/kg po), a σ1R antagonist, were administered for 4 wk (once daily) starting from the onset of aortic banding after OVX. We also examined antihypertrophic effects of pentazocine (0.5-1 μM) in cultured cardiomyocytes exposed to angiotensin II. Pentazocine administration significantly inhibited PO-induced cardiac hypertrophy and rescued hypertrophy-induced impairment of cardiac dysfunctions such as left ventricular end-diastolic pressure, left ventricular developed pressure, and left ventricular contraction and relaxation (±dp/dt) rates. Coadministration of NE-100 with pentazocine eliminated pentazocine-induced amelioration of heart dysfunction. Interestingly, pentazocine administration inhibited PO-induced σ1R reduction and inositol-1,4,5-trisphosphate (IP3) receptor type 2 (IP3R2) upregulation in heart. Therefore, the reduced mitochondrial ATP production following PO was restored by pentazocine administration. Furthermore, we found that σ1R binds to the ryanodine receptor (RyR) in addition to IP3 receptor (IP3R) in cardiomyocytes. The σ1R/RyR complexes were decreased following OVX-PO and restored by pentazocine administration. We noticed that pentazocine inhibits the ryanodine-induced Ca(2+) release from sarcoplasmic reticulum (SR) in cultured cardiomyocytes. Taken together, the stimulation of σ1R by pentazocine rescues cardiac dysfunction by restoring IP3R-mediated mitochondrial ATP production and by suppressing RyR-mediated Ca(2+) leak from SR in cardiomyocytes.
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http://dx.doi.org/10.1152/ajpheart.00300.2013DOI Listing
October 2013

Crucial interactions between selective serotonin uptake inhibitors and sigma-1 receptor in heart failure.

J Pharmacol Sci 2013 22;121(3):177-84. Epub 2013 Feb 22.

Division of Molecular Cardiovascular Biology, Department of Pediatrics, The Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

Depression is associated with a substantial increase in the risk of developing heart failure and is independently associated with increased cardiovascular morbidity and mortality. Inversely, cardiovascular disease can lead to severe depression. Thus, therapy with selective serotonin reuptake inhibitors (SSRIs) is strongly recommended to reduce cardiovascular disease-induced morbidity and mortality. However, molecular mechanisms to support evidence-based SSRI treatment of cardiovascular disease have not been elucidated. We recently found very high expression of the sigma-1 receptor, an orphan receptor, in rat heart tissue and defined the cardiac sigma-1 receptor as a direct SSRI target in eliciting cardioprotection in both pressure overload (PO)induced and transverse aortic constriction (TAC)-induced myocardial hypertrophy models in rodents. Our findings suggest that SSRIs such as fluvoxamine protect against PO- and TAC-induced cardiac dysfunction by upregulating sigma-1 receptor expression and stimulating sigma-1 receptor-mediated Akt-eNOS signaling. Here, we discuss the association of depression and cardiovascular diseases, the protective mechanism of SSRIs in heart failure patients, and the pathophysiological relevance of sigma-1 receptors to progression of heart failure. These findings should promote development of clinical therapeutics targeting the sigma-1 receptor in cardiovascular diseases.
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http://dx.doi.org/10.1254/jphs.12r13cpDOI Listing
June 2013

Determination of the critical residues responsible for cardiac myosin binding protein C's interactions.

J Mol Cell Cardiol 2012 Dec 11;53(6):838-47. Epub 2012 Sep 11.

Despite early demonstrations of myosin binding protein C's (MyBP-C) interaction with actin, different investigators have reached different conclusions regarding the relevant and necessary domains mediating this binding. Establishing the detailed structure-function relationships is needed to fully understand cMyBP-C's ability to impact on myofilament contraction as mutations in different domains are causative for familial hypertrophic cardiomyopathy. We defined cMyBP-C's N-terminal structural domains that are necessary or sufficient to mediate interactions with actin and/or the head region of the myosin heavy chain (S2-MyHC). Using a combination of genetics and functional assays, we defined the actin binding site(s) present in cMyBP-C. We confirmed that cMyBP-C's C1 and m domains productively interact with actin, while S2-MyHC interactions are restricted to the m domain. Using residue-specific mutagenesis, we identified the critical actin binding residues and distinguished them from the residues that were critical for S2-MyHC binding. To validate the structural and functional significance of these residues, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes (NRC) using cMyBP-C siRNA, and replaced the endogenous cMyBP-C with normal or actin binding-ablated cMyBP-C. Replacement with actin binding-ablated cMyBP-C showed that the mutated protein did not incorporate into the sarcomere normally. Residues responsible for actin and S2-MyHC binding are partially present in overlapping domains but are unique. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for the formation and/or maintenance of normal cMyBP-C sarcomeric distribution.
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http://dx.doi.org/10.1016/j.yjmcc.2012.08.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3496057PMC
December 2012

Targeting sigma-1 receptor signaling by endogenous ligands for cardioprotection.

Expert Opin Ther Targets 2011 Feb 5;15(2):145-55. Epub 2011 Jan 5.

Tohoku University, Graduate School of Pharmaceutical Sciences, Department of Pharmacology, Aramaki-Aoba Aoba-ku, Sendai 980-8578, Japan.

Introduction: The sigma receptors, initially described as a subtype of opioid receptors, are now considered to be a unique receptor expressed in neonatal rat cardiomyocytes and in the plasma membrane of adult rat cardiomyocytes. A number of sigma receptor ligands influence cardiovascular function and the heart has binding sites for sigma receptor ligands that alter contractility both in vivo and in vitro. The human sigma-1 receptor gene contains a steroid-binding component and gonadal steroid dehydroepiandrosterone (DHEA) which interacts with the sigma-1 receptor.

Areas Covered: We recently documented that the pathophysiological role of the sigma-1 receptor in the heart and its modulation using DHEA, was cardioprotective. Moreover, agonist-induced activation of the sigma-1 receptor modulates diverse ion channels and thereby regulates heart function. Novel concepts for understanding the pathophysiological relevance of sigma-1 receptors in the progression of heart failure, and developing clinical therapeutics targeting for the receptor in cardiovascular diseases are discussed.

Expert Opinion: Future studies should attempt to develop cardiac-specific knockdown of the sigma-1 receptor to observe its downstream signaling. We expect that these observations will lead to a novel therapeutic target for which a new class of antihypertrophic drugs can be designed.
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http://dx.doi.org/10.1517/14728222.2011.546350DOI Listing
February 2011
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