Environmental Endocrine Disrupting Chemicals and Cardiac Arrhythmogenesis

Home , Bisphenol S, Mycoestrogen

A dissertation submitted to the

Division of Research and Advanced Studies of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

in the Department of Pharmacology and Cell Biophysics of the College of Medicine

Xiaoqian Gao

B.S. Sichuan University Huaxi Medical Center, 2009

Committee Chairperson: Hong-Sheng Wang, Ph.D.

Abstract

Environmental endocrine disrupting chemicals (EDCs) are a group of exogenous

compounds that may interfere with the functioning of endogenous systems and affect human

health. Bisphenol A (BPA) is one of most ubiquitous EDCs in the manufacturing industry as a

plasticizing agent used in polycarbonate plastics and epoxy resins. It is well-documented that

human exposure to BPA is extremely wide spread. It was demonstrated that BPA, at

human-exposure relevant doses, rapidly promoted cardiac arrhythmias in female rat hearts.

However, the molecular mechanisms underlying BPA’s pro-arrhythmic effects remain unclear.

As a result of banning BPA’s use in various consumer products, bisphenol S (BPS) is

increasingly used as a substitute agent for BPA. Human populations are reported to be widely

exposed to BPS, but the biological activities and potential toxic effects of BPS are not well

understood.

The objective of this dissertation is to investigate the cardiac impact of EDCs including

BPA and BPS, with a focus on their cardiac arrhythmogenesis and underlying cellular and

molecular mechanisms. Of particular interest, was to elucidate the signaling cascades and protein targets underlying BPA’s rapid alteration of myocyte Ca2+ handling and promotion of

arrhythmogenic-triggered activities in female rodent hearts; and to evaluate how BPS affects

cardiac arrhythmogenesis in comparison to BPA.

It was demonstrated that protein kinase A (PKA) and Ca2+/Calmodulin-dependent protein

kinase II (CAMKII) signaling pathways are the two major signaling pathways activated by

BPA. In isolated female rat ventricular myocytes, BPA exposure rapidly increased

ii phosphorylation of the ryanodine receptors by PKA but not by CAMKII. BPA exposure also rapidly increased the phosphorylation of phospholamban by CAMKII but not PKA. These two pathways are mediated by estrogen receptor β but not estrogen receptor α, and are shown to be localized. Functional analysis also showed that both PKA and CAMKII were necessary contributors to the arrhythmogenesis of BPA on cardiomyocytes. This study identified the unique signaling cascades of BPA in the heart, and elucidated its novel effects on key Ca2+ handling proteins.

Also of interest is the cardiac impact of BPS, especially on the electrical aspect of the heart. It was shown that in female rat hearts, BPS rapidly increased heart rate and promoted ventricular arrhythmias under stress conditions. BPS increased arrhythmogenic-triggered activities in isolated female myocytes via alteration of Ca2+ handling, in particular by increasing spontaneous sarco/endoplasmic reticulum Ca2+ release. BPS exposure increased phosphorylation of two key Ca2+ handling proteins, the ryanodine receptor and phospholamban. Additionally, the pro-arrhythmic effects of BPS were demonstrated to be female-specific, characterized by an inverted-U dose response curve. These results provide important mechanistic insights into the rapid cardiac arrhythmogenesis of BPS in female hearts, and contribute to the evaluation of the potential cardiac toxicity of BPS.

Furthermore, the cardiac effects of probenecid were investigated. Collaboratively, it was shown that probenecid increased myocardial contractility using in vivo echocardiography, ex vivo Langendorff perfused heart and isolated myocyte system. The inotropic effect is likely mediated by transient receptor potential vanilloid 2 channels via enhanced sarco/endoplasmic reticulum Ca2+ release.

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Acknowledgments

First and foremost, I would like to express my deepest gratitude to my thesis advisor, Dr.

Hong-Sheng Wang, for being a tremendous mentor for me. Throughout my graduate studies, his careful direction and unwavering support helped me to grow, both academically and personally. His keen sense of science and dedication to research will always influence me in my future career. I am sincerely grateful for his invaluable guidance along this journey.

I want to extend my appreciation to the members of my dissertation committee, Drs. Jo

El Schultz, Terry Kirley and Steven Kleene, for their insightful advices, constructive criticism and continuous support during my training process. Their constant help with my research and my own progress is indispensible to the completion of this dissertation.

My thanks also go to the past and current members of Dr. Hong-Sheng Wang’s laboratory, who are just like my family. I would like to thank Min Dong, Weizhong Song and

Sujuan Yan for teaching me key techniques in my projects, Yamei Chen for performing myocyte contractility measurements, Qian Liang and Jianyong Ma for collaborating with me and helping me with troubleshooting, Paul Niklewski for valuable discussions and suggestions. It has been such a pleasure to work with them for all these years, and our friendship will last forever. Special thanks to our collaborator Dr. Jack Rubinstein for his insights and collaborative work in the dissertation.

I would like to thank all the faculty and students in the Department of Pharmacology for giving me so much help during my graduate studies. I am truly indebted to Dr. Jo El Schultz, who was our graduate program director, for her constant attention to my study progress and

concern for my personal life. Many thanks to our program coordinator Nancy Thyberg, for always being there to render kind help. I want to thank Janet Manning and Chi Keung Lam for being great teachers for me during my rotations in the first year, and my classmate

Clifford Cookman for constant encouragement and support.

Finally, my thanks go to my family for their love and continuous support, which have given me strength to overcome the obstacles in both studies and in life. I want to show my appreciation to my parents, for always loving me and believing in me. Especially, I would

like to thank my husband Teng, for his unconditional love and being an advocate for every little progress I made. This dissertation would not be completed without his patient help in

revision. Lastly, I want to offer gratitude to my baby Aiden, for cheering me up with his beautiful smiles in a tough day, and for helping me to understand the meaning of life.

Table of Content

Page

Abstract ...... ii

Acknowledgments ...... v

Table of Content ...... vii

List of Abbreviations ...... xi

List of Tables and Figures ...... xv

Chapter I: Introduction ...... 1

1. Environmental endocrine disrupting chemicals ...... 1

2. Bisphenol A ...... 5

3. Bisphenol A and cardiovascular diseases ...... 8

4. Cardiac conduction system and ventricular action potential (AP) ...... 10

5. Ventricular arrhythmias and cellular mechanisms ...... 13

5.1. Premature ventricular beats ...... 13

5.2. Ventricular tachycardia ...... 14

5.3. Ventricular fibrillation ...... 16

5.4. Mechanisms of ventricular arrhythmias ...... 17

5.4.1 Triggered activities: Early after-depolarization and delayed

after-depolarization ...... 17

5.4.2 Reentry arrhythmias ...... 18

5.5. Myocyte Ca2+ handling and excitation-contraction coupling ...... 20

6. Bisphenol A and cardiac arrhythmias ...... 22

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6.1. Experimental evidence ...... 22

6.2. Potential mechanisms ...... 24

7. Bisphenol S ...... 27

8. Probenecid ...... 29

9. Dissertation focus and hypotheses ...... 31

Chapter II: Materials and Methods ...... 35

1. Animals ...... 35

2. Cell dissociation and culture ...... 35

2.1. Isolation of mouse ventricular myocytes ...... 36

2.2. Isolation and culture of rat ventricular myocytes ...... 36

3. Electrophysiology recording ...... 37

3.1. Surface electrocardiography ...... 37

3.2 Transmembrane Ca2+ current recordings ...... 38

4. Evaluation of myocyte mechanical function ...... 39

4.1. Myocyte shortening measurement ...... 39

4.2. Myocyte after-contraction measurement ...... 39

5. Evaluation of myocyte Ca2+ kinetics ...... 40

5.1. Ca2+ transient measurement ...... 40

5.2. Ca2+ spark measurement ...... 41

5.3. Ca2+ after-transient measurement ...... 42

6. Immunoblotting ...... 42

6.1. General protocol ...... 42

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6.1.1. Total protein extraction from cardiomyocytes ...... 42

6.1.2. Protein concentration measurement ...... 43

6.1.3. Polyacrylamide gel running and membrane transfer ...... 43

6.1.4. Blocking, washing and incubation with antibodies ...... 44

6.1.5. Detection ...... 44

6.1.6. Stripping ...... 47

6.2. Specified protocols for proteins of interest ...... 47

7. Intracellular cAMP measurement ...... 47

8. Determine the estrous cycle phases of rats...... 48

9. Statistical analysis ...... 48

Chapter III: Results and Discussions ...... 50

Result 1. Rapid arrhythmogenic action of bisphenol A in female rat hearts and

underlying molecular mechanisms ...... 50

1.1. Background and rationale ...... 50

1.2. BPA’s rapid effects on female rat cardiac myocytes ...... 52

1.3. cAMP/PKA signaling pathway in BPA’s rapid effects ...... 55

1.4. CAMKII signaling pathway in BPA’s rapid effects ...... 58

1.5. Roles of estrogen receptors and ERK1/2 in BPA’s rapid effects ...... 61

1.6. Functional endpoints analysis: roles of PKA and CAMKII ...... 63

1.6.1. SR Ca2+ release/leak ...... 63

1.6.2. Arrhythmogenic-triggered activity ...... 66

1.6.3. Myocyte contractility ...... 66

Discussion 1 ...... 66

Result 2. Bisphenol S, a manufacturing substitute for bisphenol A, shows rapid

pro-arrhythmic effects in female rat hearts ...... 76

2.1. Background and rationale ...... 76

2.2. BPS promotes development of ventricular arrhythmias in female rat hearts .....78

2.3. BPS rapidly promoted development of arrhythmogenic-triggered activities in

female rat ventricular myocytes ...... 81

2.4. BPS rapidly affected Ca2+ handling in female rat ventricular myocytes ...... 81

2.5. BPS rapidly affected Ca2+ handling proteins in female rat ventricular myocytes

...... 84

2.6. BPS gender specificity in cardiac actions ...... 87

Discussion 2 ...... 93

Result 3. Probenecid alters mouse cardiac myocytes contractility by affecting Ca2+

handling ...... 100

3.1. Background and rationale ...... 100

3.2. Probenecid increased isolated mouse ventricular myocyte contractility in a

dose-dependent manner ...... 101

3.3. Probenecid increased cytosolic Ca2+ in isolated mouse ventricular myocytes 103

Discussion 3 ...... 107

Chapter IV: Conclusions and Significance ...... 112

Chapter V: References ...... 120

Appendix: Publications and Abstracts ...... 146

List of Abbreviations

AP Action potential

ARs Androgen receptors

AV Atrioventricular

BME β-mercaptoethanol

CASQ Calsequestrin

ChOL Chitosan oligosaccharide lactate

CICR Ca2+-induced Ca2+ release

DAD Delayed after-depolarization

DDT Dichlorodiphenyltrichloroethane

DES Diethylstilbestrol

DHPR Dihydropyridine receptors

EAD Early after-depolarization

EC coupling Excitation-contraction coupling

ECG Electrocardiography

EDCs Endocrine disrupting chemicals

EF Ejection fraction

EPA Environmental Protection Agency

EREs Estrogen response elements

ERK Extracellular signal-regulated kinases

ERs Estrogen receptors

GRs Glucocorticoid receptors

HET Heterozygous

HGFs Human gingival fibroblasts

hNav1.5 Human cardiac sodium channel

HRV Heart rate variability

2+ ICa Ca current

2+ ICaL L-type Ca current

+ INa Na current

IKr Rapid delayed rectifier K+ current

IKs Slow delayed rectifier K+ current

IK1 Inward rectifier K+ current

Iti Transient inward current

Ito Transient outward K+ current

KHB Krebs–Henseleit buffer

KO Knockout

LOAEL Lowest observed adverse effect level

LTCC Cardiac L-type Ca2+ channel

MAPK Mitogen-activated protein kinase

Maxi-K Large conductance Ca2+/voltage-sensitive K channel

MVT Monomorphic ventricular tachycardia

NCX Na+- Ca2+ exchanger

NHANES National Health and Nutrition Examination Survey

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NMDR Non-monotonic dose response

NSVT Nonsustained ventricular tachycardia

NTP US National Toxicology Program

OAT Organic anion transporter

PBBs Polybrominated biphenyls

PBS Phosphate buffered saline

PCBs Polychlorinated biphenyls

PLN Phospholamban

PPARs Peroxisome proliferator-activated receptors

PVBs Premature ventricular beats

PVCs Premature ventricular contractions

PVT Polymorphic ventricular tachycardia

RT-PCR Reverse transcription-polymerase chain reaction

RyR Ryanodine Receptor

SA Sinoatrial

SEM Standard error of the mean

SERCA Sarco/endoplasmic reticulum Ca2+ ATPase

SR Sarco/endoplasmic reticulum

TBBPA Tetrabromobisphenol A

TRs Thyroid hormone receptors

TRPV2 Transient receptor potential vanilloid 2

VEBs Ventricular ectopic beats

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VES Ventricular extrasystole

VF Ventricular fibrillation

VGCC Voltage-gated Ca2+ channels

VT Ventricular tachycardia

WT Wild type

xiv

List of Tables and Figures

Page

Tables

Table 1. Conditions for immunoblotting ...... 45

Table 2. Antibody information ...... 46

Figures

Figure I-1. Cardiac electrical conduction system and the action potential in human ventricular

mycoytes...... 12

Figure I-2. Example electrocardiography recordings of ventricular arrhythmias...... 15

Figure I-3. Early afterdepolarizations and delayed afterdepolarizations (EADs and DADs)

...... 19

Figure I-4. Illustration of myocyte Ca2+ handling and excitation-contraction coupling...... 21

Figure 1. BPA’s rapid effects in female rat ventricular myocytes ...... 53

Figure 2. BPA’s rapid effects on triggered activities in female rat myocytes from different stages of estrous cycle ...... 54

Figure 3. BPA’s rapid impact on RyR in isolated female rat ventricular myocytes ...... 56

Figure 4. BPA’s rapid impact on myocyte RyR involved cAMP/PKA signaling ...... 57

Figure 5. BPA’s rapid impact on PLN in isolated female rat ventricular myocytes ...... 59

Figure 6. BPA’s rapid impact on myocyte PLN involved CAMKII signaling ...... 60

Figure 7. Role of phospholipase C/inositol 1,4,5-trisphosphate receptor in the rapid effects of

BPA on female rat ventricular myocytes...... 62

Figure 8. Role of ERs and ERK1/2 in the rapid effects of BPA on female rat ventricular

myocytes ...... 64

Figure 9. SR Ca2+ leak in female rat ventricular myocytes under BPA exposure was mediated

by PKA but not CAMKII ...... 65

Figure 10. PKA and CAMKII’s roles in mediating BPA’s action on triggered activity ...... 67

Figure 11. PKA and CAMKII’s roles in mediating BPA’s action on myocyte contractility 68

Figure 12. Illustration of BPA’s rapid signaling cascade in female rat ventricular myocytes 70

Figure 13. Structures of bisphenol S and bisphenol A (BPS and BPA) ...... 77

Figure 14. Acute BPS exposure increased heart rate in female rat hearts ...... 79

Figure 15. Acute BPS exposure promoted ventricular arrhythmias in female rat hearts ...... 80

Figure 16. Acute exposure to BPS induced triggered activities in female rat ventricular

myocytes ...... 82

Figure 17. BPS rapidly altered Ca2+ transient in female rat ventricular myocytes ...... 83

Figure 18. BPS rapidly altered Ca2+ spark in female rat ventricular myocytes ...... 85

Figure 19. BPS rapidly altered contractility of female rat ventricular myocytes ...... 86

Figure 20. BPS rapidly increased RyR and PLN phosphorylation in female rat myocytes ....88

Figure 21. BPS increased RyR and PLN phosphorylation through ERβ ...... 89

Figure 22. BPS gender specificity in cardiac arrhythmogenesis in male rat hearts ...... 90

Figure 23. BPS gender specificity in cardiac Ca2+ handling in male rat ventricular myocytes

...... 91

Figure 24. BPS gender specificity in phosphorylation status of RyR and PLN ...... 92

Figure 25. Roles of ERα and ERβ in BPS gender specificity in male rat ventricular myocytes

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...... 94

Figure 26. Effect of probenecid on contractility of isolated mouse ventricular myocytes ...102

Figure 27. Blockade effect of ruthenium red on probenecid’s alteration of myocyte contractility ...... 104

Figure 28. Effect of probenecid on Ca2+ transient in isolated mouse ventricular myocytes 105

Figure 29. Effect of probenecid on Ca2+ spark in isolated mouse ventricular myocytes ...... 106

Figure 30. Effect of probenecid on cytosolic Ca2+ level in isolated mouse ventricular myocytes ...... 108

Figure 31. Probenecid’s effect on cytosolic Ca2+ was caused by SR Ca2+ release ...... 109

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Chapter I: Introduction

The central focus of my dissertation work is to investigate the role of environmental

chemicals in cardiac arrhythmogenesis and to understand the underlying cellular and

molecular mechanisms. With the wide spread use of environmental chemicals as a result of

modern industrialization, knowledge on how these chemicals could potentially affect human

health is critical. In addition, I also investigated the cardiac effects of a FDA-approved drug

probenecid, and examined how it could modulate myocardial function.

1. Environmental endocrine disrupting chemicals

Environmental endocrine disrupting chemicals (EDCs) are exogenous compounds that may interfere with the endogenous endocrine system and affect the reproduction and health of humans and wildlife (Diamanti-Kandarakis, Bourguignon et al. 2009). The majority of EDCs

come from synthetic chemicals used in the manufacturing industry as solvents, lubricants, plastics, thermal papers, and pharmaceutical agents. Examples of the EDCs include bisphenol

A, phthalates, polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), dichlorodiphenyltrichloroethane (DDT) and diethylstilbestrol (DES). Natural substances in plants and animals can also be EDCs, such as phytoestrogens (Diamanti-Kandarakis,

Bourguignon et al. 2009). EDCs often contain a phenolic moiety which mimics the structure of endogenous steroids and enables their binding to steroid hormone receptors. There is an increasing concern over EDCs, especially synthetic EDCs, due to their mass industrial production globally. They are dispersed into our environment and many do not decay easily due to their designed chemical stability. Even EDCs that could be degraded rapidly may also

affect living organisms under their key developmental stages. As a result, animals and

humans have been highly exposed to EDCs (see review (Colborn, vom Saal et al. 1993)).

Understanding the biological activities and health impacts of EDCs is critical for health

professionals to prevent possible adverse influences.

US Environmental Protection Agency (EPA) summarized the actions of EDCs as

“interferes with synthesis, secretion, transport, metabolism, binding action, or elimination of

natural blood-borne hormones that are present in the body and are responsible for

homeostasis, reproduction, and developmental process” (Diamanti-Kandarakis, Bourguignon et al. 2009). Endocrine disruption has been linked to a spectrum of reproductive problems in females including reduced fecundity, longer time to conceive and higher rates of miscarriage; while in males, reduced sperm count and activity have been reported (Bay, Asklund et al.

2006; Crain, Janssen et al. 2008). Studies have shown that exposure to EDCs at critical

developmental periods may result in reproductive diseases in women such as ovulation and

lactation disorders, uterine fibroids, endometriosis and breast cancer (Fenton 2006;

McLachlan, Simpson et al. 2006). One of the first observations of EDCs affecting female reproductive system was for the synthetic estrogen, DES. In 1971, adenocarcinoma of the vagina was reported in women born to mothers taking DES during the first trimester (Herbst,

Ulfelder et al. 1971). In males, disruption of prenatal testicular development by EDCs is associated with cryptorchidism, spermatogenesis defects, hypospadias, as well as prostate and testis cancers in adults (Bay, Asklund et al. 2006). Later, EDCs have also been linked to thyroid dysfunction, metabolic and cardiovascular diseases, immune diseases and neurodevelopment disorders (Casals-Casas and Desvergne 2011; Schug, Janesick et al. 2011;

Andra and Makris 2012; Frye, Bo et al. 2012).

The primary effects of EDCs are mediated by receptors. Early studies demonstrated that

EDCs could act on nuclear steroid receptors including estrogen receptors (ERs), progesterone receptors, androgen receptors (ARs), thyroid hormone receptors (TRs), glucocorticoid receptors (GRs) and peroxisome proliferator-activated receptors (PPARs). Inappropriate activation or antagonism of these receptors alters downstream gene expression, resulting in reproductive and developmental defects (Diamanti-Kandarakis, Bourguignon et al. 2009).

Later researchers found that EDCs have a much wider range of actions. EDCs could also interact with non-nuclear receptors (membrane-associated receptors) and induce non-genomic signaling, disrupt steroid biosynthesis and metabolism, as well as activate transgenerational epigenetic modifications, including DNA methylation and histone acetylation (Fisher 2004;

Tabb and Blumberg 2006; Rissman and Adli 2014).

Notably, many EDCs have low-dose effects. Generally, low-dose effects of EDCs refer to their effects that occur within the human exposure range, or at doses lower than those used in toxicological studies for traditional risk assessment (Richter, Birnbaum et al. 2007;

Vandenberg, Colborn et al. 2012). Therefore, studies focusing on low-dose effects of EDCs are more physiologically relevant to the general human population. The US National

Toxicology Program (NTP) defined BPA’s “low-dose” as doses < 50 mg/kg/day, which was also the lowest observed adverse effect level (LOAEL) identified by traditional toxicological studies. For in vivo studies, laboratory animals are orally delivered BPA ≤ 50 mg/kg animal weight/day. In in vitro tissue and cell studies, the equivalent concentration was defined as ≤ 1

× 10−7 M by the Chapel Hill expert panel, which has been widely adopted in the field.

(Wetherill, Akingbemi et al. 2007)

EDCs generate bi-phasic dose response curves as measured by many different endpoints.

These curves are also named U-shaped or inverted U-shaped non-monotonic dose response

(NMDR) curves (Vandenberg, Colborn et al. 2012). The classic view of toxicology is “the dose makes the poison”, which means higher doses of a drug are more toxic than the lower doses. Most drugs’ safety profiles were established based on this notion (Vandenberg,

Colborn et al. 2012). A monotonic dose response curve was created for the drug from measurements at high and moderate doses, and extrapolated downward to determine the safe dose (Vandenberg 2014). However, for EDCs, simply making predictions based on the lower dose as the safe dose may not be appropriate, because the lower dose could also alter biological and physiological endpoints with a high efficacy. There are still questions though, regarding the significance and cross species application, about EDCs non-monotonic dose responses (Rhomberg and Goodman 2012). The mechanisms behind EDCs’ non-monotonic dose responses have been increasingly studied and a diverse array of possible mechanisms has been proposed. For instance, decreased receptor selectivity, receptor desensitization, receptor down-regulation and cytotoxicity could lead to decreased cellular responses to EDCs at higher dose. In addition, opposing effects generated by different subtypes of receptors

(ERα and ERβ) or different subpopulations of cells could also cause U-shaped curves. These mechanisms have extended our understandings for EDCs’ non-monotonic dose responses at the cellular as well as molecular levels (see review (Vandenberg, Colborn et al. 2012)).

However, due to the diversity of EDCs and specificity of their actions in different systems and tissues, the mechanisms underlying many types of non-monotonic dose responses of

EDCs still need to be further defined.

2. Bisphenol A

Bisphenol A (BPA, CAS# 80-05-7), is a synthetic chemical first created in 1891 by

Russian chemist Aleksandr Dianin (Dodds and Lawson 1936). The manufacturing industry

started utilizing BPA in the early 1950s for producing polycarbonate plastics and epoxy resins

(Halden 2010). Ever since then, BPA is one of the largest produced industrial chemicals worldwide, exceeding 2.2 million metric tons a year. BPA enables the products made with it to be tough, water-resistant and versatile, and therefore it is incorporated in a variety of consumer products such as baby bottles, food containers, and beverage/food can linings; as well as for industrial uses such as water pipes (Halden 2010). As a result of the wide usage,

BPA has become a common environmental chemical. In the synthesis process of BPA, the reaction of polymerization is not complete, so non-polymerized monomer BPA molecules are also left in the products. Over time, these BPA monomers can be released into food or beverage, especially under harsh environmental conditions such as exposure to heat, acid or base (Brotons, Olea-Serrano et al. 1995; Kang, Kito et al. 2003; Le, Carlson et al. 2008). It is also known that human exposure of BPA occurs through inhalation as well (Wilson, Chuang et al. 2007). Industrial synthesis alone is estimated to release up to 100 tons of BPA into the air per year (Vandenberg, Maffini et al. 2009). It is shown by multiple studies that BPA is at detectable levels in human biological fluids in over 90% of individuals tested in different populations. The mean or median BPA concentrations in urine were in the low μg/L (nM) range, as reported by various human exposure assessment studies (Calafat, Kuklenyik et al.

2005; Itoh, Iwasaki et al. 2007; Cantonwine, Meeker et al. 2010; Ning, Bi et al. 2011; Kim

and Park 2013). Human studies have shown that after oral administration, BPA (low-dose

with 5 mg/person) is rapidly absorbed via the gastrointestinal tract; the maximal plasma

concentration is reached at around 80 minutes following BPA intake. Glucuronidation of BPA

in the liver is the major way of metabolism. The formed BPA glucuronide conjugate is rapidly

cleared from blood through urinary excretion, with a half-life of 6 hours; elimination is essentially complete with 24 hours after oral administration.

BPA belongs to the family of endocrine disrupting chemicals (EDCs). Early studies in

1930s identified estrogenic activities of BPA (Dodds and Lawson 1936). Later, BPA was shown to be able to bind to estrogen receptors and interfere with endogenous estrogens in various in vitro cell lines (Gould, Leonard et al. 1998; Pennie, Aldridge et al. 1998; Vivacqua,

Recchia et al. 2003; Recchia, Vivacqua et al. 2004). BPA is known to exert its effects by binding and activating the nuclear steroid receptors ERα and ERβ, which translocate into the

nucleus and interact with estrogen response elements (EREs) to modify the expression of

estrogen-responsive genes (Klinge 2001). Recruitment of different co-regulatory factors

associated with EREs enables the regulation of a wide range of target genes (Klinge 2000;

Klinge 2001). In addition, BPA binds to androgen receptors and exhibits anti-androgenic

effects in vitro (Sohoni and Sumpter 1998). BPA is also shown to bind to thyroid hormone

receptors and act as an antagonist (Moriyama, Tagami et al. 2002).

Besides the classic genomic signaling through steroid nuclear-receptors, BPA was shown

to directly induce intracellular signaling transduction pathways. This activation of

non-genomic signaling by BPA is through mechanisms independent of the gene transcription

regulatory activity of nuclear hormone receptors (Wetherill, Akingbemi et al. 2007). Cell

signaling cascades are able to amplify the signal from very low doses of ligand to impact its target proteins and alter the function of the cell rapidly. BPA’s rapid signaling has been

reported in a variety of systems including pancreas, pituitary, breast cancer, cerebellar cortex

as well as the heart. (Quesada, Fuentes et al. 2002; Walsh, Dockery et al. 2005; Wozniak,

Bulayeva et al. 2005; Zsarnovszky, Le et al. 2005; Gao, Liang et al. 2013). These rapid

effects of BPA exposure are observed within seconds to minutes, and the underlying mechanisms are believed to involve receptors with extracellular accessible binding sites as

well as intracellular targets to modulate intracellular signaling, such as membrane associated

estrogen receptors (Watson, Campbell et al. 1999). It is known that BPA can activate

MAPK/ERK signaling, PKC signaling, PKA signaling and PKG signaling in a tissue-specific

manner (Canesi, Lorusso et al. 2004; Alonso-Magdalena, Laribi et al. 2005; Belcher, Le et al.

2005). Further studies are still needed to understand the complete rapid signaling cascades

behind BPA’s rapid effects in the cells.

Studies have shown that acute BPA exposure occurs in people’s daily life. Carwile’s

colleagues recruited volunteers from Harvard School of Public Health students and staff, who

were assigned to consume a 12-ounce serving of fresh soup or canned soup daily for 5 days.

It was found that canned soup daily consumption was associated with a significant increase in

urinary BPA (> 10-fold) (Carwile, Ye et al. 2011). The interior epoxy resin coatings of the

cans prevent metal corrosion, while at the same time release BPA into people’s diet. Another

study by Tateoka showed that following consumption of canned coffee drink, BPA

concentration in the breast milk was about 5 times higher than before the coffee intake. And

the peak BPA concentration was detected at 1 hour in the breast milk (Tateoka 2014). So far,

the impact of acute BPA exposure on the human health was still not well understood. This

dissertation focuses on the acute effects of BPA on the cardiovascular system, especially the

electrical activity of the heart, which provides knowledge on how the acute increase of BPA

in the body affects the function of endogenous systems.

3. Bisphenol A and cardiovascular diseases

There have been extensive studies to examine the impact of BPA on human health.

Potential links between BPA exposure and human diseases have been reported, including cancer, obesity, diabetes and disorders of the reproductive, neuroendocrine and immune systems (Diamanti-Kandarakis, Bourguignon et al. 2009; Zoeller, Brown et al. 2012).

Recently, epidemiological studies have identified the cardiovascular system as another potential target of BPA.

NHANES, or National Health and Nutrition Examination Survey, is a program of studies to analyze the status of health and nutrition of US populations. Total (free and conjugated)

BPA levels were measured in the urine samples of participants. Lang’s colleagues first reported an association between higher urinary BPA level and cardiovascular diseases based on the 2003-2004 NHANES data. In 1,455 adult participants, it was found that the adjusted mean urinary BPA levels were higher in participants diagnosed with cardiovascular diseases including coronary artery disease, angina and heart attacks, and statistically significant association was identified between higher BPA concentrations and cardiovascular diseases and diabetes (Lang, Galloway et al. 2008). The same research group later analyzed the data from the 2005-2006 NHANES, and showed that higher urinary BPA concentrations were still significantly associated with a diagnosis of coronary artery disease, but not angina or

myocardial infarction. The difference was likely caused by a 30% decrease in urinary BPA

concentrations detected in 2005-2006 NHANES participants, which decreased the statistical

power to detect significant associations. In support of this notion, when pooled 2003-2004

and 2005-2006 NHANES data was analyzed, it was found that higher BPA urine

concentrations were significantly associated with all three diagnoses of cardiovascular

diseases (angina, myocardial infarction and coronary artery disease) (Melzer, Rice et al.

2010).

In another longitudinal study, Melzer’s group evaluated BPA’s impact on the

development of coronary artery disease by following participant’s disease conditions for 10

years. The authors demonstrated that higher urinary BPA was associated with the incidence of

coronary artery disease diagnosis (Melzer, Osborne et al. 2012). Later, it was identified that

higher urinary BPA level was also associated with angiography defined coronary

atherosclerosis in patients, which was suggested to be the causal link between high urinary

BPA levels and coronary artery diseases (Melzer, Gates et al. 2012). In addition, higher BPA exposure was also shown to be associated with peripheral arterial disease (Shankar, Teppala et al. 2012), hypertension (Shankar and Teppala 2012) and heart rate variability (HRV) (Bae,

Kim et al. 2012).

However, a study by LaKind’s investigators noticed that NHANES data may not be

sufficient to prove the association between BPA exposure and cardiovascular diseases. Their

analysis discovered inconsistencies in several previous studies based on NHANES data in

terms of methods and definitions. They used a consistent method to analyze four NHANES

data sets, and showed that urinary BPA levels in NHANES participants were not significantly

associated with coronary artery disease, heart attack or diabetes (LaKind, Goodman et al.

2012). They stated that the fact that different methodologies could lead to inconsistent

outcomes brought up questions on how reliable NHANES was to establish associations or

causal relationships between urinary BPA levels and cardiovascular diseases. They suggested that caution should be used in drawing conclusions using NHANES data (Lakind, Goodman et al. 2014). Moreover, Olsen’s colleagues did not find any associations between BPA exposure and cardiovascular diseases risk factors in a cross-sectional study (Olsen, Lind et al.

2012).

Nevertheless, some epidemiological studies, both cross-sectional and longitudinal, showed that BPA exposure is associated with cardiovascular diseases or their risk factors in adults. These include several independent analyses of NHANES data to demonstrate the association between higher urinary BPA concentrations and diagnosis of cardiovascular diseases in participants. However in other studies, no association was found between BPA exposure and cardiovascular diseases. These findings highlight the need for further assessment of BPA’s impact on the cardiovascular system and underlying mechanisms. In this dissertation, I focused on BPA’s effects on cardiac arrhythmia endpoint and investigated the underlying molecular mechanisms.

4. Cardiac conduction system and ventricular action potential (AP)

As a muscular organ, the heart functions as a pump to deliver blood throughout the body.

The electrical conduction system in the heart is a network of nodes and pathways conducting

electrical signals, allowing efficient and rhythmic contraction of the heart, which is critical

for the continuous supply of oxygen and nutrients for all the organs. The electrical stimulus

originates from the sinoatrial (SA) node located in the right atrium, travels through the atria

to the atrioventricular (AV) node located in the interatrial septum, inducing the contraction of

both atria. Three internodal pathways conducted the electrical signal transduction between SA

node and AV node. After a delay, the signal goes through the Bundle of His, which then splits

into the left and right bundle branches in the interventricular septum, activating the

contraction of the left and right ventricles respectively. These Two bundle branches taper out

to produce specialized Purkinje fibers, which are responsible for stimulating the contraction

of individual groups of myocytes (Figure I-1A) (Christoffels and Moorman 2009).

From myocyte to myocyte, the electrical signals are conducted through gap junctions.

The stimulation of a myocyte initiates a series of actions involving the influx and efflux of

various types of cations and anions, which together create an action potential (AP) in the

myocyte. In the ventricular myocytes from humans, the AP waveform is characterized by a

spike-and-dome morphology (Magyar, Kiper et al. 2004). There are 5 phases in this type of

AP (Figure I-1B). When a myocyte is electrically stimulated, Na+ channels are rapidly

+ activated, resulting in an inward Na current (INa) which depolarizes the membrane and

produces the phase 0 upstroke. Then the Na+ channel quickly inactivates, and the transient outward K+ channel activates. The transient outward K+ current (Ito) is the predominant

repolarizing current in the early phase of repolarization, producing a phase 1 notch. In the

2+ phase 2, L-type Ca current (ICaL) depolarizes the cell, which was balanced with the rapid and slow delayed rectifier K+ currents (IKr and IKs), forming the phase 2 plateau. During the

+ phase 3, ICaL inactivates, while IKr and IKs are joined by the inward rectifier K current (IK1)

which repolarize the myocyte membrane to the resting potential. IK1 remains the open state

Figure I-1. Cardiac electrical conduction system and the action potential in human

ventricular myocytes. A is adapted from Human Anatomy & Physiology 5th edition,

Benjamin Cummings, San Francisco, 2001. B is modified from (Pourrier, Schram et al. 2003)

in phase 4, which contributes to setting the resting membrane potential (Kleber and Rudy

2004).

5. Ventricular arrhythmias and cellular mechanisms

Arrhythmias are caused by irregular electrical activities of the heart. Ventricular

arrhythmias originate from the ventricles, which incorporates a series of abnormal cardiac

rhythms, including premature ventricular beats (PVBs), monomorphic and polymorphic

ventricular tachycardia (VT) as well as ventricular fibrillation (VF). It is known that ventricular arrhythmias, especially sustained VT and VF, are important causes of morbidity in cardiac disease patients; they are also the most common causes of sudden cardiac death with

75% - 80% among all cases (Olshausen, Witt et al. 1991). Ventricular arrhythmias are not only found in patients with structural cardiac diseases, but also seen in people showing no demonstrable heart diseases (Goel, Srivathsan et al. 2013).

5.1. Premature ventricular beats

Premature ventricular beats (PVBs), also known as premature ventricular contractions or complexes (PVCs), ventricular ectopic beats (VEBs) or ventricular extrasystole (VES), are

early depolarizations initiated from the ventricle due to enhanced automaticity. For diagnosis,

electrocardiography (ECG) needs to be performed on the patients experiencing palpitations.

Holter monitoring can be used for continuous recordings to increase the chance of recording

the events. PVBs can be identified from ECG graphs as the extra waves between normal ones

(Figure I-2A). Their characteristic properties are: 1) changed morphology of QRS and T

waves, 2) spacing to preceding wave is shorter, while to proceeding wave is longer than

normal, 3) proceeding waves are not affected. Based on the number within each PVBs, these

extra waves are named as bigeminy, trigeminy, and quadrigeminy. More than 3 PVBs

occurring in a row is called ventricular tachycardia (Ng 2006).

PVBs are considered benign events and are usually not associated with clinical

significance in healthy people. However, there are four major concerns regarding the frequent

development of PVBs. First, PVBs are often associated with monomorphic VT, which increases the risk for developing sustained VT (Sheldon, Gard et al. 2010). Second, combined with the existence of enhanced automaticity and polymorphic VT, PVBs may couple with the preceding QRS complex, triggering ventricular fibrillation (Sheldon, Gard et al. 2010). Third, it is found that PVBs have adverse impacts on cardiac function and can lead to cardiomyopathy, which is named PVB-induced cardiomyopathy (Chugh, Shen et al. 2000). It was shown that suppression of frequent PVBs in patients with idiopathic dilated cardiomyopathy improved left ventricular systolic function. Most patients in this study did not have underlying structural cardiac disease, but developed dysfunction of ventricles and

dilated cardiomyopathy as a result of frequent PVBs (Duffee, Shen et al. 1998). Fourth, with

the existence of impaired ventricular function due to cardiac defects or cardiac diseases,

PVBs are even deleterious to cardiac function (Lee, Klarich et al. 2012). Therefore, PVBs can

be used as a primary indicator for cardiac arrhythmogenesis and potential cardiac toxicity

caused by exogenous chemicals.

5.2. Ventricular tachycardia

Nonsustained ventricular tachycardia (NSVT) is a series of consecutive PVBs, usually

more than three, occurring at a rate greater than 150 beats per minute, but lasting less than

30 seconds (Figure I-2B). Similar to PVBs, NSVT are only transient and are mostly

Figure I-2. Example electrocardiography recordings of ventricular arrhythmias. A.

Premature ventricular beats. B. Non-sustained ventricular tachycardia. C. Sustained

ventricular tachycardia. D. Ventricular fibrillation. (Adapted from MEDLIBES online

medical library, website: http://medlibes.com/)

well-tolerated causing no severe symptoms (Sheldon, Gard et al. 2010). NSVT are generally

not associated with mortality but could affect the prognosis of patients with underlying

cardiovascular diseases (Sheldon, Gard et al. 2010). Sustained ventricular tachycardia (VT)

refers to ventricular rhythm at a rate of higher than 150 beats per minute, and lasting for more

than 30 seconds (Figure I-2C). On ECG recordings, they are characterized by wide complex

tachycardia (QRS duration > 140 msec) (Goel, Srivathsan et al. 2013). Sustained VT can be

fatal and it mostly occurs with cardiovascular diseases including coronary artery disease,

congenital cardiac diseases, dilated cardiomyopathy as well as valve diseases. There is also

idiopathic VT found mostly in young patients with no indication of structural heart diseases

(Goel, Srivathsan et al. 2013). According to the ECG morphology, VT can be classified into

two categories: monomorphic ventricular tachycardia (MVT) and polymorphic ventricular

tachycardia (PVT) (Ulus, Kudaiberdieva et al. 2013). For MVT, though different from the

baseline sinus rhythm, QRS wave morphology and length of cycle are uniform during the

arrhythmic periods. PVT is more unstable, varying continuously in QRS wave morphology

and/or cycle length (Ulus, Kudaiberdieva et al. 2013).

5.3. Ventricular fibrillation

Ventricular fibrillation (VF) is turbulent electrical activity of the heart, in which the heart

rate is higher than 350 beats per minute, so myocardial pumping functioning is largely

impaired due to uncoordinated contractions (Figure I-2D). As a result, blood pressure is

significantly dropped, effective blood circulation is ceased and oxygen cannot be delivered to

vital organs (Jalife 2000). On the ECG recordings, VF can be diagnosed from the aperiodic,

ever-changing ventricular electrical complexes. VF is always a medical emergency requiring

immediate medical care, and remains a major cause for sudden cardiac death. Development

of VF is often associated with cardiac diseases such ischemic heart disease, and also observed

with disturbed electrolyte homeostasis and overdosing cardiotoxic drugs. Idiopathic VF could

be found in patients with no heart diseases diagnosed as well (Jalife 2000).

5.4. Mechanisms of ventricular arrhythmias

5.4.1. Triggered activities: Early after-depolarization and delayed after-depolarization

Triggered activities are myocyte electrical activities “triggered” by the preceding impulse.

They are not initiated from the nodal pacemaker cells (Antzelevitch and Burashnikov 2011).

In 1995, Pogwizd demonstrated in a rabbit heart failure model that spontaneous occurrences

of ventricular arrhythmias such as PVBs and VTs were initiated by nonreentrant mechanisms,

i.e., triggered activities (Pogwizd 1995). There are two types of triggered activities classified

based on their relative timing: early after-depolarization (EAD) and delayed

after-depolarization (DAD). EAD interferes with action potential repolarization phases

including phase 2 and phase 3, while DAD occurs after full repolarization is completed.

When the amplitude of EAD or DAD is sufficient to depolarize the myocyte membrane to the

threshold potential, a spontaneous triggered action potential will follow the preceding

one(Pogwizd and Bers 2004). These triggered activities lead to ventricular extrasystoles in

the heart and increase cardiac arrhythmogenesis (Antzelevitch and Burashnikov 2011).

Occurrence of EAD involves the imbalance between repolarizing currents and

2+ depolarizing currents. Increased inward late sodium current (late INa), Ca current (ICa) or sodium-calcium exchanger (NCX) current, as well as decreased outward currents such as rapid delayed rectifier K+ current and slow delayed rectifier K+ current (IKr and IKs) all

contribute to the prolonged repolarization phase of the action potential (Figure I-3). As a

2+ result, phase 2 and phase 3 prolongation allow L-type Ca current (ICaL) to reactivate and

depolarize the membrane, generating a second action potential upstroke (January and Riddle

1989).

DAD is caused by elevated intracellular Ca2+ load in the myocytes. Spontaneous

sarcoplasmic reticulum (SR) Ca2+ release (or SR leak) and augmented [Ca2+]i activate the

forward mode of NCX (Ca2+ efflux) and generate a net transient inward current (Iti), resulting

in depolarization of myocyte membrane and triggering a new action potential (Pogwizd and

Bers 2004). So different from EADs, DADs are observed after full repolarization of

preceding action potentials (Figure I-3). DADs are known to occur under rapid stimulation

rates, such as in the presence of β-adrenergic receptor stimulation (Pogwizd and Bers 2004).

Experimentally, following repeated high-frequency electrical pacing, myocytes with altered

Ca2+ handling can possibly develop DADs during the resting phase as a result of increased

SR leak or [Ca2+]i overload. These DADs could be captured as spontaneous excitation

activities of myocytes, such as after-contractions and after-Ca2+ transients (Yan, Chen et al.

2011).

5.4.2. Reentry arrhythmias

Reentry arrhythmias are based on a “circuit” movement of cardiac electricity, which is

mechanistically different from automaticity or triggered activity (Ebinger, Krishnan et al.

2005). Under the normal condition, myocardium is not susceptible to rapid cardiac rhythm

because it is protected by high electrical conduction velocity and a long refractory period.

However, with pathological conditions present such as myocardial infarction, there is an

Figure I-3. Early afterdepolarizations and delayed afterdepolarizations (EADs and

DADs). AP, action potential. Arrow indicates action potential duration prolongation.

(Adapted from Pogwizd and Bers, 2004)

abnormally slow conducting area and a unidirectional blocking of the propagation of action

potential (el-Sherif, Gough et al. 1985). In addition, for the “circuit” movement to continue,

the propagation time around the loop must be long enough to allow recovery of each site in

the loop. When the reentry circuit is formed, it will repetitively reinforce the electrical

conduction and induce ventricular arrhythmias (Antzelevitch and Burashnikov 2011).

5.5. Myocyte Ca2+ handling and excitation-contraction coupling

Ca2+ is a major intracellular messenger with vital roles in many cellular responses. In

cardiomyocytes, Ca2+ transmits the electrical signal to the sarcomere, thereby triggering a

mechanical event. The systolic-diastolic cycle via synchronized cellular depolarization and

subsequent activation of contractile proteins is called excitation-contraction coupling (EC

coupling) (Bers 2008). To facilitate this process, the total intracellular concentration of Ca2+,

and more importantly, local concentrations within defined spatial regions of the cell, are

tightly regulated via specific Ca2+ channels and binding proteins. Voltage-gated Ca2+ channels

(VGCCs) located in the T-tubules, also named L-type Ca2+ channels (LTCCs) or

dihydropyridine receptors (DHPRs), mediate Ca2+ entry upon plasma membrane depolarization above a threshold potential during an action potential. T-tubules are deep sarcolemma invaginations, adjacent to the specialized Ca2+ storage compartment named sarcoplasmic reticulum (SR) (Figure I-4). This Ca2+ influx into the cytosol stimulates Ca2+

release from SR, via ryanodine receptors (RyRs) located on the surface of SR membranes.

The process is called Ca2+-induced Ca2+ release (CICR). Ca2+ molecules subsequently bind to

the contractile protein troponin C, causing a conformational change of the associated troponin and tropomyosin complex. This exposes the binding site for myosin on actin, and a sliding

Figure I-4. Illustration of myocyte Ca2+ handling and excitation-contraction coupling.

Modified from (Bers 2002).

motion is generated, ultimately resulting in the contraction of myocytes. During diastole,

Ca2+ is cleared from the cytosol by reuptake into SR under the action of sarcoplasmic

reticulum Ca2+ ATPase (SERCA), which is regulated by phospholamban (PLN), and

transported out of the sarcolemma through NCX (Bers 2008). EC coupling is a critical

process to maintain the normal electrical and mechanical functioning of cardiomyocytes. Ca2+

cycling has been intensively studied and much evidence has been obtained that disorders

within the cycling pathway may cause arrhythmogenic-triggered activities such as DADs.

Alterations of RyR function, for example, are shown to be associated with cardiac

arrhythmias (Wehrens and Marks 2003).

RyR is activated when Ca2+ binds to its sensor subunit, resulting in opening of the RyR

channel and a large amount of SR Ca2+ release (Van Petegem 2012). [Ca2+]i is readily increased from 150 nM at diastole to over 1 μM at systole, which is referred to as a Ca2+

transient (Guatimosim, Dilly et al. 2002). During the diastolic state of myocytes, an efflux of

Ca2+ through RyR was described by Cheng’s colleagues. This transient and local SR Ca2+ release can be observed by confocal microscopic line scanning as Ca2+ sparks (Cheng,

Lederer et al. 1993). These sparks are generated by synchronous activation of multiple RyR

channels, in the form of clusters each composed of 6 to 20 RyRs. So a Ca2+ spark is

considered the fundamental unit of SR Ca2+ release, and the Ca2+ transient can be described

as a large number of sparks generated coordinately (Guo, Zhang et al. 2006).

6. Bisphenol A and cardiac arrhythmias

6.1. Experimental evidence

Previously, our laboratory investigated the rapid impact of low-dose BPA on cardiac

electrical rhythm using adult rat hearts. It was demonstrated by surface ECG recordings that

under catecholamine-induced stress condition, exposure to 10-9 M BPA rapidly and

significantly increased ectopic ventricular beat frequency in ex vivo hearts from female, but

not male adult rats. And at the cellular level, rapid exposure to 10-9 M BPA induced the

development of “triggered activities” in female but not male ventricular myocytes (Yan, Chen

et al. 2011). In myocytes, triggered activities are aberrant spontaneous excitations. They are

regarded as one of the important mechanisms for arrhythmogenesis (Bers 2002). The rapid

effects of BPA exposure on cardiac arrhythmogenesis following ischemia injury were

subsequently examined in our laboratory (Yan, Song et al. 2013). Ischemia injury is a

common complication during myocardial infarction, and it may result in damage to the

myocytes and dysfunction of myocardium. It is also known that ischemia injury can cause

acute arrhythmias in the heart and sudden cardiac death. It was shown that exposure to 10-9 M

BPA at reperfusion following ischemia increased sustained ventricular arrhythmias duration

in female, but not male ex vivo hearts (Balke, Kaplinsky et al. 1981; Bernier, Manning et al.

1989). So it was demonstrated that low-dose BPA has cardiac pro-arrhythmic effects in

female rodent hearts, which were significant under pathophysiological conditions such as

stress and ischemia.

Recently, a study by Posnack’s group systematically examined BPA’s rapid effects on

cardiac electrophysiology using ex vivo adult rat hearts. After 15 minutes of BPA treatment,

cardiac electrical activities of the adult rat hearts were recorded via ECG and optical mapping.

It was shown that in ex vivo female rat hearts, 10-6 to 10-4 M BPA prolonged the PR segment

on ECG recordings, increased action potential duration and decreased ventricular conduction

velocity. BPA exposure resulted in 3rd degree atrioventricular block at the highest dose of

10-4 M (Posnack, Jaimes et al. 2014). Authors did not provide direct evidence on the

mechanisms underlying observed electrical alteration in the heart, but suggested that BPA’s

actions on cardiac sodium channels, Maxi-K channels and L-type Ca2+ channels were all

potential contributors to the observed myocardial electrical conduction dysfunction.

6.2. Potential mechanisms

Underlying cellular mechanisms of the rapid cardiac arrhythmogenesis of BPA were

investigated by our laboratory. It was shown by Yan’s investigators that BPA’s pro-arrhythmic effects on female rat myocytes were mediated by rapid alteration of myocyte Ca2+ handling

(Yan, Chen et al. 2011). Ca2+ handling is a key process in myocyte physiology, mediating the coupling of electrical excitation and mechanical contraction. Abnormal Ca2+ handling could

lead to cardiac arrhythmogenesis (Bers 2002; Bers 2008). It was shown that exposure to 10-9

M BPA rapidly increased sarcoplasmic reticulum (SR) Ca2+ release, Ca2+ reuptake and SR

Ca2+ stores in female rat ventricular myocytes on a beat-to-beat basis. Notably, SR “Ca2+ leak”, which is the diastolic spontaneous SR Ca2+ release, was significantly increased upon

BPA exposure; and blocking aberrant SR Ca2+ release completely abolished BPA’s pro-arrhythmic effects in the myocytes (Yan, Chen et al. 2011). It was also shown that the gender specificity of BPA’s cardiac effects was caused by the counterbalancing actions of

ERα and ERβ. Myocyte mechanical contraction was used to evaluate the process of Ca2+

handling. ERβ activation increased myocyte contraction in both female and male rat

myocytes, while ERα activation decreased myocyte contraction in both genders. Because the

combined effects of ERα and ERβ determined the response of myocytes to BPA, in females,

the stimulatory ERβ signaling was dominant; while in males the inhibitory ERα signaling

counterbalanced ERβ resulting in no detectable responses. Indeed, ablation of ERβ in mice or

using an ERβ blocker abolished the arrhythmogenic effects of BPA in female myocytes. And

blockade of ERα revealed the stimulatory effects of ERβ in male myocytes under BPA

exposure, resulting in a female-like response (Yan, Chen et al. 2011; Belcher, Chen et al.

2012).

Equally important was the assessment of non-monotonic dose responses of BPA. Our

laboratory demonstrated that over the dose range of 10-12 to 10-6 M, BPA had an inverted-U shaped dose response in female rat ventricular myocytes, measured by multiple endpoints including myocyte contraction, Ca2+ transient amplitude, and triggered activities development.

We found that the most efficacious dose of BPA in myocytes was 10-9 to 10-8 M, which overlapped with the reported exposure levels for humans (Liang, Gao et al. 2014). The mechanisms behind the non-monotonicity of BPA’s cardiac effects were further investigated.

It was shown that the inverted-U shaped dose responses were created based on BPA’s multiple monotonic actions on individual myocyte Ca2+ handling processes. BPA increased

SR Ca2+ leak and Ca2+ reuptake with monotonicity, and also suppressed L-type Ca2+ current

with a monotonic dose response. Reduction in Ca2+ influx at high (micromolar) doses

accounts for the decline phase in the inverted-U shape dose responses (Liang, Gao et al.

2014).

Systemic molecular mechanistic studies are still lacking, though some studies have

provided important evidence to suggest the involvement of ion channels and Ca2+ handling

proteins. Asano’s investigators showed that BPA (10-5 to 10-4) activated Maxi-K channels,

which are the large conductance Ca2+/voltage-sensitive K+ channels in coronary artery

smooth muscle cells from both dogs and humans, in a reversible, dose-dependent,

non-genomic manner (Asano, Tune et al. 2010). In a heterologous cell system transiently

expressing human cardiac sodium channels (hNav1.5), O’Reilly’s colleagues demonstrated

-5 that BPA inhibited hNav1.5 current with a EC50 of 2.5 × 10 M. Steady state inactivation of

-4 hNav1.5 shifted to more hyperpolarized potentials upon 10 M BPA exposure, suggesting that

BPA binds to inactivate-state channels (O'Reilly, Eberhardt et al. 2012). Deutschmann et al.

reported the inhibition of L-type Ca2+ channels by BPA in mouse ventricular myocytes with an

-5 2+ EC50 of 2.5 × 10 M. This study also evaluated a group of other voltage-gated Ca channels

and identified that the N-type, P/Q-type, and T-type Ca2+ channels could be inhibited by BPA

with EC50 values in the micromolar range (Deutschmann, Hans et al. 2013). A more recent

study by Michaela’s group showed that BPA inhibited three subtypes of T-type Ca2+ channels which were expressed in HEK293 cells with different potencies. While low-dose (nM) BPA

2+ inhibited T-type Ca channels with an order of potency of: Cav3.2 ≥ Cav3.1 > Cav3.3;

high-dose (μM) BPA’s inhibition potency was Cav3.3 ≥ Cav3.2 > Cav3.1 (Michaela, Maria et

al. 2014). Nonetheless, these studies mostly used high-dose BPA (> 10-7) and may not directly

reflect the low-dose BPA cardiac arrhythmogenic actions.

Patel’s colleagues showed that on the protein expression levels, male mice showed

increased expressions in sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), sodium-calcium exchanger (NCX) and calsequestrin (CASQ) following chronic BPA exposure, which resulted in increased intracellular Ca2+ removal and SR Ca2+ storage. In

female mice, phospholamban (PLN) expression was significantly increased with BPA

treatment, likely leading to reduced SR Ca2+ reuptake and Ca2+ load (Patel, Raad et al. 2013).

However, authors focused on the cardiac concentric remodeling but not arrhythmia analyses

in this study. And their results were different from our laboratory’s Ca2+ handling studies on

isolated rat ventricular myocytes (Yan, Chen et al. 2011), which was likely due to the chronic

BPA exposure in their experimental design and subsequent compensatory mechanisms.

A significant knowledge gap exists in the molecular mechanisms through which

low-dose BPA rapidly promotes cardiac arrhythmogenesis, limiting our understanding of the

consequences of wide spread BPA exposure, particularly on the cardiovascular system. In this

dissertation, the myocyte-specific signaling mechanism underlying low-dose BPA’s acute

arrhythmogenic action will be investigated, with a focus on alterations on the proteins

involved in Ca2+ handling and arrhythmogenic-triggered activities.

7. Bisphenol S

Bisphenol S (BPS, 4,4’-Sulfonyldiphenol, CAS# 80-09-1) is another member of the bisphenol family, composed of two phenol groups on each side of a sulfonyl group (Chen, Ike et al. 2002). Similar in its structure to BPA, BPS is being used as a substitute plasticizing agent in many “BPA-free” products. BPS is also a component of thermal papers used as receipts, envelopes and boarding passes (Liao, Liu et al. 2012). It is reported that even under normal usage, BPS still leaches from food cans and containers (Vinas, Campillo et al. 2010).

Recent study has showed that BPS is now becoming a wide-spread environmental chemical and can be detected in human samples (Liao and Kannan 2013). BPS was found in 81% of urine samples collected from US populations as well as seven Asian countries at a mean concentration of 0.65 ng/mL (2.6 nM) (Liao, Liu et al. 2012).

Though limited, an increasing number of studies suggest that BPS also has estrogenic

endocrine disrupting activities, like its analogue BPA. In various heterologous cell systems,

BPS was shown to have genomic estrogenic activities. Most of these studies used doses

higher than those detected in humans, from μM to mM (Hashimoto, Moriguchi et al. 2001;

Kuruto-Niwa, Nozawa et al. 2005; Grignard, Lapenna et al. 2012). At human-exposure

relevant doses (nM), BPS was shown to adversely affect reproduction and progeny

generation of zebrafish (Ji, Hong et al. 2013). It also significantly affected the steroid

hormone synthesis process of human adrenal cortico-carcinoma cells, by altering the

production of progesterone, testosterone and cortisol (Rosenmai, Dybdahl et al. 2014).

Additionally, BPS has also been shown to activate rapid signaling processes. Vinas’ group

demonstrated that acute exposure to low-dose BPS activated ERK signaling in a pituitary cell

line (Vinas and Watson 2013).

Compared to BPA, BPS was shown to have similar dose response curves in the

activation of estrogen receptors in MCF7 cells (Kuruto-Niwa, Nozawa et al. 2005); but

Molina-Molina’s investigators recently demonstrated that BPS was less potent than BPA in

binding to ER and ER in competitive receptor binding assays, and also less potent in activating ER’s transcriptional activity as measured in a luciferase reporter gene system

(Molina-Molina, Amaya et al. 2013). BPS was also found to have anti-androgen activities. Its potency to bind and activate androgen receptors was less than BPA (Kitamura, Suzuki et al.

2005).

Though still controversial, BPA has been linked to cardiovascular diseases and potential risk factors by multiple epidemiological studies (Lang, Galloway et al. 2008; Melzer, Rice et

al. 2010; Melzer, Osborne et al. 2012; Shankar, Teppala et al. 2012). Previously, our

laboratory also demonstrated the ability of low-dose BPA to rapidly promote

arrhythmogenic-triggered activities in female rat ventricular myocytes (Yan, Chen et al.

2011). Moreover, under stress or ischemia, BPA increased cardiac arrhythmic events in the

female rat hearts (Yan, Chen et al. 2011; Yan, Song et al. 2013). The potential adverse impact

of BPA on the heart was demonstrated by these studies. However, the biological activities of

BPS on the heart and its potential cardiac toxicity are completely unknown. This poses a

major gap in our knowledge on the health impact of EDCs, especially those BPA substitute

chemicals used widely in the consumer market as a new BPA replacement. In this

dissertation, the rapid action of low-dose BPS on rat hearts as well as isolated

cardiomyocytes was investigated, with a focus on the arrhythmogenesis of BPS and

underlying cellular and molecular mechanisms.

8. Probenecid

This dissertation focuses on the EDCs and how they impact cardiac function, particularly

on cardiac arrhythmogenesis. In addition, the dissertation addresses the cardiac effects of

probenecid, with a focus on its action on the mechanical function of cardiomyocytes.

Probenecid is a FDA-approved drug primarily used for treating hyperuricemia and gout

(Robbins, Koch et al. 2012). It was initially developed to decrease the renal excretion, and

increase the serum level of antibiotics such as penicillin, referred to as “Benemid”(Boger,

Pitts et al. 1950). Probenecid was administered to patients through slow intravenous infusion,

which could cause local irritation. Later, probenecid was found to be rapidly absorbed with

oral administration, reaching peak serum concentrations within 1 to 5 hours. Currently in the

US, probenecid’s lipid soluble preparation for oral administration is available (Boger, Beatty et al. 1950).

In early studies of probenecid, its uricosuric effect was discovered and its clinical use was extended to gout treatment (Robbins, Koch et al. 2012). The mechanism involves inhibition of organic acid reabsorption, such as uric acid, in the renal proximal tube.

Probenecid inhibits the organic anion transporter (OAT) and prevents reuptake of uric acid

back to the blood (Roch-Ramel and Guisan 1999). Although used as the standard treatment

for gout, with new therapies showing better efficacy, probenecid’s clinical use has declined

significantly. Probenecid was also shown to inhibit active transport processes in other

systems including the eye, brain and liver (Forbes and Becker 1960; Korf and van Praag 1970;

Kenwright and Levi 1973), but these effects have not resulted in clinical uses yet.

Recently, probenecid was identified to be a selective agonist of transient receptor potential vanilloid 2 (TRPV 2) channels (Bang, Kim et al. 2007). In neurology, transient receptor potential (TRP) channels have been extensively studied; they are involved in transduction of temperature responses in sensory ganglia neurons (Vriens, Nilius et al. 2014).

However, the function of TRP channels in the cardiovascular system was not clear, only a few

members in the TRP family were shown to have direct effects in the heart, such as

TRPC3/6/7 in cardiac hypertrophy development triggered by pressure overload (Wu, Eder et

al. 2010). Within the TRPV subfamily, TRPV2 has been shown to have direct cardiac actions.

Iwata’s investigators demonstrated that cardiac-specific overexpression of TRPV2 led to

dilations of all chambers in the mouse heart (Iwata, Katanosaka et al. 2003). Yet the possible

effects of probenecid on cardiomyocytes were never investigated. In this dissertation,

probenecid’s cardiac actions and how it could modulate myocardial function were examined,

with a focus on its alteration on the mechanical function of cardiomyocytes.

9. Dissertation focus and hypotheses

The overall objective of this dissertation research is to investigate the impact of

environmental endocrine disrupting chemicals on the heart with a focus on cardiac

arrhythmogenesis and its underlying cellular and molecular mechanisms. The dissertation evaluated the acute effects of two environmental chemicals, BPA and BPS, on the rodent hearts and isolated cardiomyocytes, focusing on the electrophysiological properties; and examined alterations in cardiomyocyte Ca2+ kinetics and signaling cascades upon rapid

exposure.

9.1. Hypothesis 1: The rapid impact of BPA on myocyte arrhythmogenesis is mediated

by PKA and CAMKII signaling pathways.

Previous work in our laboratory showed that BPA at human-exposure relevant doses (nM)

rapidly promoted arrhythmogenic-triggered activities in cardiac myocytes from female rat

hearts (Yan, Chen et al. 2011). Under conditions including catecholamine-induced stress and

ischemia, BPA induced arrhythmia development in ex vivo female rat hearts (Yan, Chen et al.

2011; Yan, Song et al. 2013). BPA’s cardiac arrhythmogenic effects were likely mediated by

ERβ signaling and through rapid alteration of myocyte Ca2+ handling, particularly enhanced

SR Ca2+ leak; yet, the molecular mechanisms were not known. In this study, our goal was to

investigate the signaling cascade by which low-dose BPA alters Ca2+ handling. This dissertation focuses on two important Ca2+ handling proteins RyR and PLN. RyR is the major intracellular Ca2+ release channel on SR, and RyR-mediated SR Ca2+ release is critical for

cardiac function (Bers 2008). Abnormal RyR Ca2+ release is known to play a key role in myocyte arrhythmogenesis (Betzenhauser and Marks 2010). PLN is a regulator of SERCA function, modulating Ca2+ reuptake. Both RyR and PLN can be phosphorylated by PKA and

CAMKII to alter their activities (Bers 2008). Immunoblotting was utilized to evaluate the

phosphorylation status of RyR and PLN in isolated ventricular myocytes upon acute low-dose

BPA exposure. Confocal microscopic line-scan imaging was used to measure SR Ca2+ leak

and triggered activities in myocytes when BPA exposure is antagonized with PKA and

CAMKII inhibitors.

9.2. Hypothesis 2: Bisphenol S, a substitute for Bisphenol A, has a rapid pro-arrhythmic

effect in female hearts.

BPS is now increasingly being used as a substitute agent for BPA by the manufacturing

industry of consumer goods and thermal papers. Like BPA, BPS is found to leach from food

containers (Vinas, Campillo et al. 2010). Liao’s colleagues reported that human exposure to

BPS was wide spread; nM BPS was detected in urine samples collected from populations in

eight countries (Liao, Liu et al. 2012). BPS was found to have estrogenic activities

(Hashimoto, Moriguchi et al. 2001; Grignard, Lapenna et al. 2012). Though the health impact

of BPA has drawn attention from government agencies and public media, currently the

knowledge on the potential toxicity of BPS is still limited. The goal of this study is to

elucidate the rapid impact of BPS in rodent hearts and its underlying mechanism. On the whole heart level, ex vivo adult rat hearts were exposed to BPS, at the same time monitored for ECG characteristics. On the myocyte level, myocyte triggered activities were recorded with confocal microscopic line-scan imaging upon exposure to BPS. Ca2+ handling proteins

RyR and PLN phosphorylation levels were measured using immunoblotting from myocytes

treated with BPS. Both female and male adult rat hearts were tested for gender differences.

9.3. In addition to the central objective of the dissertation, the cardiac effects of probenecid on isolated cardiomyocytes were investigated, with the hypothesis that probenecid regulates cardiomyocyte mechanical function.

The studies describing the effect of probenecid on cardiac function are very limited. In

1950s, Bronsky’s investigators reported that probenecid (benemid) used by congestive heart failure patients showed a strong diuretic effect, but they attributed it to probenecid’s renal action instead of any potential cardiac action (Bronsky, Dubin et al. 1955). Erttmann’s group investigated the effects of probenecid in isolated atrial tissues from guinea pigs and showed that probenecid increased its steady-state contractility in a dose-dependent manner; yet the mechanism for this cardiac effect was not discussed (Erttmann 1978). Our collaborator, Dr.

Rubinstein, performed expression tests for TRPV channels on murine and human myocardial tissue, and confirmed the expression of TRPV2, which was especially abundant in the left ventricle (Koch, Gao et al. 2012). These findings led us to investigate the cardiac effects of probenecid from the whole animal, isolated heart, and isolated cardiomyocyte levels. Data from Dr. Rubinstein’s lab showed that probenecid increased myocardial contractility on both in vivo heart and isolated heart from adult mice (Koch, Gao et al. 2012). To determine the effects of probenecid on the myocyte level, fractional shortening was measured to evaluate the mechanical function of isolated mouse ventricular myocytes upon exposure to probenecid.

A pharmacological inhibitor for TRPV2 channels, ruthenium red, was used to evaluate the target for probenecid on the myocytes. [Ca2+]i was measured via confocal microscopic

fluorescent imaging on the myocytes to study possible cellular mechanisms.

Chapter II: Materials and Methods

1. Animals

All animal procedures were performed following protocols approved by the University of

Cincinnati Institutional Animal Care and Use Committee and in accordance to

recommendations of the Panel on Euthanasia of the American Veterinary Medical

Association. Adult C57BL6J mice (10 – 12 weeks) and Sprague-Dawley rats (175 – 199g) were used in the studies as non-surviving sources of hearts and ventricular myocytes. Sodium pentobarbital (80 mg/kg, intraperitoneal) was used for euthanization of animals. Animals were housed in standard polycarbonate caging maintained on a 14 h light, 10 h dark light cycle. Irradiated aspen sani-chip bedding (P.J. Murphy Forest Products Corp. Montville, NJ) was required to prevent the exposure of possible corn-based mycoestrogen. Animals were fed ad libitum Teklad diet 2020 (Harlan Laboratories Inc.) free of soybean meal, alfalfa and animal products which could possibly introduce uncontrolled levels of estrogenic chemicals to the experimental animals. Animals’ drinking water was filtered by a specialized water purification system (Millipore Rios 16 with ELIX UV/Progard 2) which could reduce oxidizable organics to less than 1% of initial source levels. Glass bottles were used to dispense drinking water to animals. It was confirmed that the concentrations of BPA in drinking water as well as experimental reagents were under the detection threshold of a sensitive BPA-specific ELISA assay (minimum detection limit = 0.05 ng/mL) (Le, Carlson et al. 2008).

2. Cell dissociation and culture

2.1. Isolation of mouse ventricular myocytes

Adult C57BL6J mice (10 – 12 weeks) of both sexes were anaesthetized by intraperitoneal injection of sodium pentobarbital. Mouse heart was quickly excised and rinsed to get rid of blood. Then the heart was mounted on a Langendorff perfusion system (driven by pump) and perfused with a modified oxygenated Ca2+-free Krebs–Henseleit buffer (KHB)

composed of (mM) NaCl 118, KCl 5.4, HEPES 10, NaH2PO4 0.33, MgCl2 2, glucose 10, taurine 30 and butanedione monoxime 10 (pH=7.4, circulating water bath 38.5 °C). After the blood was cleared, an enzyme solution (KHB containing 0.8 mg/mL type II collagenase,

0.1% BSA and 25 μM CaCl2) was used to digest the heart for total 10 minutes until the heart became flaccid. The ventricles of heart were excised, minced, pipette-triturated and filtered in

KHB solution. After sufficient sedimentation (no centrifugation), the supernatant was

removed and the myocytes were resuspended in KHB solution. Ca2+ concentration in KHB was gradually raised to 1.8 mM with 0.1 M CaCl2. Isolated myocytes were stored at room temperature (25 °C) and used within 4 hours on the day of isolation.

2.2. Isolation and culture of rat ventricular myocytes

Adult Sprague-Dawley rats (175 – 199g) of both sexes were anaesthetized by intraperitioneal injection of sodium pentobarbital. Rat heart was excised and cannulated through the aorta quickly, then mounted on a Langendorff perfusion system (gravity driven) perfused with modified KHB composed of (mM) NaCl 118, KCl 4.8, HEPES 25, K2HPO4

1.25, MgSO4 1.25, glucose 11, taurine 30 and butanedione monoxime 10 (pH=7.4, circulating

water bath 37.8 °C). The KHB solution was oxygenated during perfusion. When the blood

was cleared, the heart was perfused with an enzyme solution containing 0.7mg/mL

collagenase type II (298 U/mg), 0.2 mg/mL hyaluronidase, 0.1% BSA and 25 μM CaCl2 for

30 minutes. The enzyme solution for digesting the heart was used repeatedly, reducing the

amount needed for a single isolation. During the digestion process, the Ca2+ concentration in

the perfusion buffer was increased to 100 μM using 0.1 M CaCl2 (add 12.5 μL and 25 μL at

10 minutes and 15 minutes, respectively). When the perfusate from the heart became a stream,

feel the heart till it turned flaccid. The heart was dismounted, and the ventricular tissue was

excised, minced and repeatedly pipetted to suspend the myocytes. Then the suspension was

filtered through gauze to remove tissue fragments. Isolated myocytes were harvested and

resuspended in KHB after being centrifuged briefly (100 x g for 1.5 minutes). Ca2+

concentration in KHB was gradually raised to 1.0 mM with 0.1 M CaCl2 for immediate

experiments. For culturing, myocytes were suspended in a Medium-199 based solution

(Gibco; catalog number 31100–035, with Earle’s salts and L-glutamine, without NaHCO3, lot

number 561846; prepared using BPA free water) containing 5 mM creatine, 2 mg/mL BSA, 5

mM taurine, 2 mM L-carnitine, 100 IU/mL penicillin as well as 100 μg/mL streptomycin, and

plated on laminin-coated polystyrene culture dishes at a density of approximately 5 x 104 cells/cm2. Myocytes were allowed to adhere and stabilize for 4 hours in a 37 °C incubator

prior to treatment.

3. Electrophysiology recording

3.1. Surface electrocardiography (surface ECG)

Adult rats were anesthetized with sodium pentobarbital. After dissection, the hearts were

rapidly cannulated via the aorta, and mounted on a Langendorff apparatus in retrograde mode

with Krebs-Henseleit solution containing (mM) NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2,

EDTA 0.5, CaCl2 2.5, NaHCO3 25, and glucose 11 (pH =7.4, bubbled with 95% O2 and 5%

CO2, circulating water bath 38.2 °C) at 80 mmHg and 15 mL/minute perfusion rate. Hearts

were perfused in control solutions for at least 1 hour to stabilize before exposure to treatment

drugs. Surface electrocardiograph was continuously measured from the heart, with two

electrodes positioned at the base and apex on the surface of the heart. Data was collected and

analyzed using the Powlab 4/30 data acquisition system and LabChart 7 software. Following

1 hour of stabilization control condition, treatment drugs were added into the perfusate and

hearts were continuously monitored for 20 minutes.

3.2. Transmembrane Ca2+ current recordings

Isolated myocytes were maintained at room temperature (25 °C) and perfused in a glass

chamber with Tyrode’s solution containing (mM) NaCl 140, KCl 5.4, MgCl2 1, CaCl2 1.8,

HEPES 5, and glucose 10 (pH = 7.4). After the membrane was successfully ruptured,

extracellular solution was switched to a Na+- and K+-free solution containing (mM): tetraethylammonium chloride (TEA-Cl) 265, CsCl 5.4, CaCl2 2, MgCl2 1, HEPES 5, glucose

10 and 4-aminopyridine 3 (pH=7.4), to eliminate contamination caused by other ionic and

exchange currents. For recording Ca2+ current in the myocytes, pipettes were filled with

solution containing (mM): aspartic acid 115, CsOH 115, CsCl 20, MgCl2 2.5, EGTA 11,

HEPES 10, Mg-ATP 2, Na-GTP 0.1 (adjusted pH = 7.2 using CsOH). Glass pipette had a

resistance of 1.5 – 2.5 MΩ. For L-type Ca2+ recordings, myocytes were clamped at –50 mV

for 5 minutes for dialysis of the intracellular solution and stabilization of Ca2+ currents before

the measurement started. Activation of the L-type Ca2+ current was achieved by depolarizing

steps from a holding potential of –50 mV to voltages ranging from –40 to +60 mV, in 10 mV

increments. To treat the myocytes, probenecid was dissolved in extracelluar Na+- and K+-free solution to perfuse the chamber. Data were collected using pCLAMP9 software through an

Axon Digidata 1322A data acquisition system.

4. Evaluation of myocyte mechanical function

4.1. Myocyte shortening measurement

Mechanical properties of isolated ventricular myocytes were assessed at room temperature (25 °C) using a video based edge-detection system (Crescent electronics, Sandy,

UT) at a 60-Hz frame rate. Mouse or rat ventricular myocytes were placed in a plexiglass

chamber mounted on the stage of an inverted microscope. The chamber was filled with the

standard Tyrode’s solution containing (mM) NaCl 140, KCl 5.4, MgCl2 1, CaCl2 1.8, HEPES

5, and glucose 10 (pH = 7.4, for rat ventricular myocytes, CaCl2 = 1.0 mM). During treatment,

the solution also contained various drugs. A CCD camera was attached to the camera port of

the inverted microscope, and the output was connected to a video-edge detector (Crescent

electronics, Sandy, UT), which monitored changes in position of both the right and left cell

edges of the rectangular myocytes. Electrical field stimulation was used to induce contraction

of myocytes with a Grass S48 stimulator (Grass instrument, Gincy, MA) with 2 ms 1.5x

threshold pulses at 0.5 Hz. Data were sampled through an Axon Digidata 1322A board using

the PCLAMP 9 software (Molecular Devices, Sunnyvale, CA). Fractional shortening was

analyzed using Clampfit 9 software to evaluate the contractility of isolated ventricular

myocytes.

4.2. Myocyte after-contraction measurement

Similar to shortening measurements, isolated rat ventricular myocytes were placed in a

plexiglass cell chamber filled with standard Tyrode’s solution containing (mM) NaCl 140,

KCl 5.4, MgCl2 1, CaCl2 1, HEPES 5, and glucose 10 (pH = 7.4, at room temperature 25 °C).

During treatment of myocytes, the solution also contained indicated drugs. Contraction of

myocytes was recorded from 2 to 7 minutes following drug treatment. Myocytes were excited

with electrical field stimulation by a Grass S48 stimulator with 2 ms 1.5x threshold pulses at

a rate of 2 Hz for 8 seconds and recorded for an additional 15 seconds to capture possible

spontaneous contractions (triggered activities). Myocyte contraction was imaged with a CCD

camera and examined using a video-edge detector. Signals were sampled through an Axon

Digidata 1322A board using the PCLAMP 9 software.

5. Evaluation of myocyte Ca2+ kinetics

5.1. Ca2+ transient measurement

To measure intracellular Ca2+, Fluo-4 acetoxymethyl (AM) ester (Molecular Probes,

Eugene, OR) was used to produce fluorescent signals. A 1.0 mM stock solution was made by

adding 45 μL of DMSO into 50 μg Fluo-4 AM in a vial protected from light exposure (MW:

1096.95). Aliquots of 10 μL were prepared from the stock solutions and wrapped with aluminum foil and stored at -20 °C. Before experiments, the stock solution was diluted to 4 –

5 μM in the culture medium. Mouse or rat ventricular myocytes were loaded in the medium solution for up to 20 minutes at room temperature (25 °C) on the rocker, or up to 10 minutes in a 37 °C incubator (myocytes may be cultured overnight for stabilization). Then myocytes were washed at least three times with medium solution to remove extracellular Fluo-4 AM, and incubated at room temperature (25 °C) for 10 minutes to allow complete hydrolysis of the Fluo-4 esters. Tyrode’s solution was used during the process of drug treatment and

measurement. Intracellular Ca2+ fluorescent signals were captured by a ratiometric

photometry system (Intracellular Imaging Inc., Cincinnati, OH) with an excitation

wavelength of 488 nm and a long pass 510 nm emission filter. Changes in intracellular Ca2+ concentration ([Ca2+]i) were calculated from the fluorescence unit ΔF/F0. By fitting the data to a single exponential decay function curve, the Ca2+ transient decay time constant value was

determined.

5.2. Ca2+ spark measurement

Isolated mouse or rat ventricular myocytes were loaded with Fluo-4 AM at 4 – 5 μM in the medium solution for up to 20 minutes at room temperature (25 °C), and then washed with medium solution at least three times to remove residual Fluo-4 AM, followed by waiting 10 minutes after washing at room temperature (25 °C) to allow complete de-esterification.

Myocytes were placed in Tyrode’s solution containing 1.8 mM CaCl2 with or without

treatment drug, in a custom made plexiglass recording chamber. Ca2+ spark was recorded in quiescent, unstimulated myocytes between 2 to 7 minutes following drug treatment (for time

course, continuously recorded) with a Zeiss LSM 710 inverted confocal microscope with a

40x water-immersion objective lens and an excitation wavelength of 488 nm. Ca2+

fluorescence emission signals were measured at 515 nm in the line scan mode with 3.07 ms

intervals, and each line comprises 512 pixels spaced at 0.056 mm. Analysis of Ca2+ spark

imaging was performed using IDL software (ITT Visual Information Solutions, Boulder, CO).

The frequency of Ca2+ spark was calculated as the number of sparks divided by the length of

scanning line, divided by the time of scanning, which was expressed as n/100 μm/sec. The

frequency of Ca2+ spark indicates the average count of sparks per spatial distance per unit

time.

5.3. Ca2+ after-transient measurement

Ca2+ after-transient was measured by confocal microscopic line scan imaging. Similar to

Ca2+ spark measurement, isolated rat ventricular myocytes were loaded with Fluo-4 AM (4 –

5 μM) in the medium solution, and then washed with medium solution at least three times.

Myocytes were placed in a custom made plexiglass recording chamber filled with Tyrode’s

solution with or without treatment drug. Myocytes were paced with electrical field stimulation via a Grass S48 stimulator with 2 ms 1.5x threshold pulses at a rate of 2 Hz.

Images were obtained at 515 nm of a Zeiss LSM 710 inverted confocal microscope in the line scan mode with an excitation wavelength of 488 nm. Recordings lasted for 8 seconds at 2 Hz, and then continued for another 15 seconds to capture possible Ca2+ after-transients (triggered

activities).

6. Immunoblotting

6.1. General protocol

6.1.1. Total protein extraction from cardiomyocytes

Isolated rat ventricular myocytes were treated with drugs for the indicated length of time,

washed with ice-cold phosphate buffered saline (PBS) solution for three times, and then

collected with 1 mL ice-cold PBS solution. Each treatment dish contained about 3 x 105 myocytes. The solution with myocytes was centrifuged and the cell pellets on the bottom were snap frozen in liquid nitrogen. Total proteins were extracted with a commercial cell lysis buffer (Cell Signaling Technology) supplemented with phosphatase inhibitor cocktail sets I and II (EMD Millipore Chemicals), and complete protease inhibitor cocktail (Roche

Applied Science). Stored myocyte pellet samples were thawed on ice and then 100-150 μL

supplemented cell lysis buffer was added, based on pellet sizes. The extraction lasted for

about 1 hour with mixing every 5 minutes. The samples were then centrifuged at 12,000 x g

for 10 minutes at 4 °C. Proteins (in the supernatant) were quickly frozen in liquid nitrogen

and stored in - 80 °C for future use.

6.1.2. Protein concentration measurement

Protein concentration measurement was performed based on the colorimetric microtiter

plate protocols from Biorad. First, the Dye Reagent Concentrate stock (containing commassie

brilliant blue G-250) was diluted with distilled water at 1:5 ratio, and filtered with a Whatman

#1 paper to remove undissolved particulates. To make a standard curve, five dilutions of stock bovine serum albumin (1 mg/mL) were prepared within the linear range of 0.05 mg/mL to 0.5 mg/mL. 10 μL protein samples and standards were added to the 96-well plate in triplicate. Diluted dye reagent of 200 μL was then added into each well of the plate. The solutions in the plate were mixed and incubated at room temperature (25 °C) for at least 5 minutes before reading. A Thermomax microplate reader (GMI, Inc., Ramsey, Minnesota) was used to measure the absorbance of the protein solutions at 595 nm.

6.1.3. Polyacrylamide gels and membrane transfer

Protein samples of desired amount (see Table 1) were mixed with loading sample buffer containing: 0.125 M Tris-HCl (pH 6.8), 20% glycerol (v/v), 4% SDS (w/v), 2%

β-mercaptoethanol (BME, v/v, add freshly), containing trace amount of Bromophenol Blue

(in water). For ERK1/2, mixed protein samples were boiled at 96 °C for 10 minutes, and then put on ice to cool down. For RyR and PLN, mixed protein samples were not boiled.

Polyacrylamide gels were made at different percentages for specific proteins (see Table 1).

Gels were run for indicated times at constant voltage of 120-150 mV for different protein

targets (see Table 1). When finished, the gel was then rinsed with PBS + 0.1% Tween 20

(PBST) and transferred to a 0.22 μm nitrocellulose membrane (Bio-rad, Hercules, CA) using

a transfer cassettes sandwiched with 2 sponges, 4 pieces of filter paper, nitrocellulose membrane and gel. The transfer was carried out at constant current according to Table 1.

6.1.4. Blocking, washing and incubation with antibodies

Following the transfer, membranes were washed with PBST on a rocker for 10 minutes, and then blocked with 5% blotting milk (in PBST) for one hour to decrease antibody non-specific binding to the membranes. The membranes were incubated overnight with primary antibodies in the cold room and secondary antibodies at room temperature (25 °C) for one hour. Both primary and secondary antibodies were diluted as detailed in Table 2 in

5% blotting milk (in PBST). After incubation, the membranes were washed with PBST (10 minutes x 3) to remove excess and unbound antibody.

6.1.5. Detection

To detect the proteins on the membranes, an enhanced chemiluminescence western blotting detection system (GE Healthcare, Buckinghamshire, UK) was used. After washing, the membranes were treated with reagents which created chemiluminescence by reacting with horseradish peroxidase conjugated to secondary antibodies. In a dark room, treated membranes were overlaid with X-ray film, which was developed to visualize the bands. The exposure time of membranes to films need to be adjusted experimentally so the intensity of bands was suitable for quantification. AlphaEaseFC software (Alpha Innotech) was used to

Table 1. Conditions for immunoblotting

Loading Target Percentage of Amount Running condition Transfer condition Protein resolving gel (μg)

150 mV/240 min 85mA/overnight RyR 50 6% (cold room) (cold room)

120 mV/90 min 240 mA/90 min PLN 25 12% (cold room) (cold room)

ERK1/2 20 15% 120 mV/120 min 200 mA/180 min

Table 2. Antibody information

Western Name of Antibody Manufacturer, catalog # Species Blot Dilution RYR2 Phospho Serine 2808 Badrilla, A010-30 Rabbit 1:5000 Anti-Serum RYR2 Phospho Serine 2814 Badrilla, A010-31 Rabbit 1:5000 Anti-Serum anti-RYR antibody ABR, MA3-916 Mouse 1:1000 Phospholamban Phospho Badrilla, A010-13 Rabbit 1:5000 Threonine-17 Anti-Serum Phospholamban Phospho Badrilla, A010-12 Rabbit 1:5000 Serine-16 Anti-Serum Anti-Phospholamban A1 Badrilla, A010-14 Mouse 1:1000 Antibody Phospho-p44/42 MAPK Cell Signaling, 4370 Rabbit 1:500 (ERK1/2) (Thr202/Tyr204) p44/42 MAPK (ERK1/2) Cell Signaling, 4695 Rabbit 1:2000 Anti-rabbit IgG, HRP linked Cell Signaling, 7074S Goat 1:2000 Antibody Anti-mouse IgG, HRP linked Cell Signaling, 7076S Horse 1:2000 Antibody

measure the band intensities. The densitometric values of treatment groups need to be normalized to control bands and expressed as fold changes.

6.1.6. Stripping

The same membranes could be reprobed with another antibody after stripping of the original antibody. Membranes were incubated in stripping buffer containing 62.5 mM Tris

(pH 6.8), 2% SDS, and 100 mM β-mercaptoethanol at 65 °C for 30 minutes. Then the membranes were washed in PBST for 5 – 6 times each for 10 minutes. Following the washes, membranes were blocked with 5% blotting milk for 30 minutes, incubated with primary antibody and secondary antibodies and prepared for detection of proteins of interest.

6.2. Specified protocols for proteins of interest

Different protein targets require specified western protocols. Table 1 lists the

immunoblotting conditions for different protein targets in the dissertation studies.

7. Intracellular cAMP measurement

Cultured rat ventricular myocytes were subjected to drug treatments in a 37°C incubator.

For each treatment group, there are typically about 3 x 105 myocytes. Termination of drug

treatment was achieved by aspirating the medium and adding 0.1 M HCl to the dishes.

Intracellular cAMP level of myocytes was determined using the Direct cAMP ELISA kit

(Enzo Life Sciences), following the instructions from manufacturers. Reactions occurred in

the 96-well plate provided in the kit. OD data were obtained using a Thermomax microplate

reader (GMI, Inc., Ramsey, Minnesota) to measure the absorbance of samples at 405 nm after

reaction. The cAMP standard curve was plotted using the four parameter sigmoidal logistic

nonlinear regression fit. Levels of cAMP in samples were calculated based on the cAMP

standard curve. Values of treatment groups were normalized to control and expressed as fold

changes relative to control.

8. Determine the estrous cycle phases of rats

Vaginal lavage method was used to collect the vaginal secretion from the female rats

(Marcondes, Bianchi et al. 2002). A sterile plastic pipette was filled with 10 μL saline

solution (0.9% NaCl) and gently inserted into the rat vagina. The vaginal fluid was collected

by repeatedly pipetting the saline solution, and placed on a glass slide for microscopic

examination. Under a light microscope with 10 and 40 x objective lenses, the morphology of

the cells in the vaginal secretion was determined. Three types of cell morphology can be

observed: big round ones are the epithelial cells, irregular-shaped ones are the cornified cells,

and small round ones are the leukocytes. The estrous cycle phases of the female rats were

determined by examining the proportion of these three types of cells (Marcondes, Bianchi et al. 2002). Proestrus phase was identified with the majority of cells (more than 75%) being

epithelial cells; estrus phase was characterized with a large amount of cells in the vaginal

secretion and majority of cells being cornified cells; and diestrus phase was mostly composed

of leukocytes.

9. Statistical analysis

All data presented were analyzed with SigmaPlot 11.0 software and expressed as the

mean value ± standard error of the mean (SEM). Statistical significance for differences in

values is considered as p < 0.05. Comparison between control group and drug treatment

group was done with a two-tailed unpaired Student’s t-test. Differences before and after drug

treatment was analyzed with a two-tailed paired Student’s t-test. Alterations identified in a

time course, in a dose range, or in multiple drug treatment groups were assessed using

one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test for

multiple comparisons. Frequency of events such as percentage of myocyte showing triggered

activities was analyzed using 2 statistical test.

Chapter III: Results and Discussions

Result 1. Rapid arrhythmogenic action of bisphenol A in female rat hearts

and underlying molecular mechanisms

1.1. Background and rationale

Bisphenol A (BPA) is one of the highest produced synthetic chemicals worldwide, with

more than 2 million metric tons of production per year. It is mostly used in the manufacturing

of polycarbonate plastics and epoxy resins. BPA has been used extensively in consumer

products such as food containers, beverage and food can lining, thermal paper, dental sealants,

as well as water pipes. It is known that there is widespread and well-documented human

exposure to BPA coming from food, drinking water, dermal exposure and household dust

inhalation. BPA has been detected in human samples such as urine and blood at nanograms

per milliliter levels in more than 90% of people examined in various sampled populations

(Calafat, Kuklenyik et al. 2005; Ye, Pierik et al. 2008; Vandenberg, Chahoud et al. 2010;

Lakind and Naiman 2011; Hoekstra and Simoneau 2013).

Being an estrogenic endocrine disruptor, BPA could affect endogenous systems through a

variety of actions. Previous studies indicated that BPA exposure can negatively impact the

human body: associations were found with cancer, obesity, diabetes, disorders of the

reproductive, neuroendocrine, and immune systems (Diamanti-Kandarakis, Bourguignon et al.

2009; Zoeller, Brown et al. 2012). The cardiovascular system is recently also recognized as a

potential target of BPA’s adverse impact. Epidemiological studies have shown that BPA

exposure was associated with cardiovascular diseases such as coronary artery disease and

peripheral arterial disease (Lang, Galloway et al. 2008; Melzer, Rice et al. 2010; Melzer,

Osborne et al. 2012; Shankar, Teppala et al. 2012). Previously, our lab demonstrated that BPA

rapidly promoted arrhythmogenic-triggered activities in cardiac myocytes from female rat

hearts. Under conditions including catecholamine-induced stress and ischemic injury, BPA

promoted cardiac arrhythmic events in female rat hearts (Yan, Chen et al. 2011; Belcher,

Chen et al. 2012; Yan, Song et al. 2013). The cellular mechanism of the pro-arrhythmic

actions of BPA was shown to involve rapid alteration of myocyte Ca2+ handling, specifically

sarcoplasmic reticulum (SR) Ca2+ release/leak and SR Ca2+ reuptake (Yan, Chen et al. 2011).

Molecular mechanisms underlying BPA’s arrhythmogenesis in the heart were not fully understood, which was regarded as one of the major knowledge gaps in exploring the

consequences of BPA exposure (Wetherill, Akingbemi et al. 2007). This dissertation sought

to elucidate the signaling pathways underlying the rapid effects of BPA on arrhythmogenesis

and alteration of Ca2+ handling in the cardiac myocytes. Ca2+ handling is a key process to

cardiac physiology. It links the electrical excitation with mechanical contraction of myocytes.

The whole process consists of several steps: Ca2+ influx via L-type Ca2+ channel, release of

Ca2+ from the SR through the ryanodine receptors (RyR), and Ca2+ removal from the cytosol

by SR Ca2+ reuptake through sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and Ca2+ extrusion through the Na+/ Ca2+ exchanger (at a much lesser extent) (Bers 2008). RyR is a

Ca2+ release channel localized on the SR in cardiac myocytes. The Ca2+ release activity of

RyR is critical for normal functioning of cardiac myocytes (Bers 2008). It is known that

aberrant RyR Ca2+ release plays a key role in cardiac arrhythmogenesis (Betzenhauser and

Marks 2010). Phospholamban (PLN) is a regulator protein to SERCA by inhibiting SERCA

Ca2+ reuptake activity (Kranias and Hajjar 2012). This dissertation investigated the signaling

mechanisms by which BPA affected myocyte Ca2+ handling, particularly on RyR and PLN,

two key Ca2+ handling proteins.

1.2. BPA’s rapid effects on female rat cardiac myocytes

Previous work by our lab has shown BPA’s female-specific rapid actions on rodent

ventricular myocytes (Yan, Chen et al. 2011; Belcher, Chen et al. 2012). In this study, the rapid effects (2–7 min) of low-dose BPA (1 nM) on isolated female rat ventricular myocytes were examined. The use of nanomolar concentrations was due to its relevance to BPA exposure levels found in human populations (Calafat, Kuklenyik et al. 2005; Vandenberg,

Chahoud et al. 2010). In Figure 1A, rapid BPA treatment increased spontaneous excitation events in female rat ventricular myocytes under repeated pacing (2 Hz), which were known

as triggered activities and key arrhythmogenic mechanisms in the heart (Bers 2008). Earlier

work in our lab indicated that BPA’s pro-arrhythmic effects in the myocytes were mediated by rapid alteration of Ca2+ handling, specifically on SR Ca2+ release/leak (Yan, Chen et al.

2011). In Figure 1B, BPA rapidly increased Ca2+ spark frequency in quiescent female rat ventricular myocytes, which represented the spontaneous SR Ca2+ release through RyRs.

Rapid BPA exposure also significantly increased contractility of female rat ventricular myocytes (Figure 1C).

To test the potential impact of animal estrous cycle on BPA’s pro-arrhythmic effects in the myocytes, we measured the spontaneous excitation events (triggered activities) in the isolated ventricular myocytes from female rats in different stages of the estrous cycle, including estrus, diestrus and proestrus (Figure 2). BPA increased the percentage of myocytes

Figure 1. BPA’s rapid effects in female rat ventricular myocytes. A, Example confocal

microscopic line scan images showing Ca2+ transients in isolated female rat ventricular

myocytes, induced by electrical pacing under control (left) and under acute BPA exposure (1 nM, right). Red arrow indicates the spontaneous Ca2+ after-transient (i.e., triggered activity)

after pacing (n = 6 hearts). In the control group, 3 out of 49 myocytes exhibited triggered

activities, and in the BPA treatment group, 16 out of 52 myocytes exhibited triggered

activities. P < 0.01. B, Representative confocal microscopic line scan images showing Ca2+

spark (indicated by the red arrow) in myocytes under control (left) and under acute BPA

exposure (1 nM, right). Ca2+ spark frequency for control group was 1.55 ± 0.18 (per 100

μm/sec) and for BPA group was 2.75 ± 0.19 (per 100 μm/sec). (n = 12 myocytes from 4

hearts for each group; P < 0.05) C, Representative contraction traces from isolated female rat

ventricular myocytes under control (left) and under acute BPA exposure (1 nM, right).

Fractional shortening (FS) for control group was 8.3% ± 0.14% (n = 49 myocytes from 4 hearts) and for BPA group was 14.1% ± 0.21% (n = 44 myocytes from 4 hearts; P < 0.05).

Control

BPA N.S.

* * *

Figure 2. BPA’s rapid effects on triggered activities in female rat myocytes from

different stages of estrous cycle. Data on percentages of myocytes showing triggered

activities under control or BPA treatment in different estrous cycle stages of the female adult

rats. n = 213 myocytes in 9 hearts. BPA = 1 nM. *, P < 0.05 vs control; N.S., not significant

between three stages.

showing triggered activities in the female rats, to a similar extent from the three stages (estrus

5.6% to 33.3% vs. diestrus 5.6% to 30.6% vs. proestrus 5.6% to 36.1%), suggesting that

endogenous estrous cycle of the female rats may not be a factor affecting BPA’s

pro-arrhythmic effects in the myocytes.

1.3. cAMP/PKA signaling pathway in BPA’s rapid effects

RyR activity is regulated by protein kinase phosphorylation, including protein kinase A

(PKA) at serine 2808 and Ca2+/Calmodulin-dependent protein kinase II (CAMKII) at serine

2814. RyR phosphorylation at either 2808 or 2814 sites increases RyR opening probability through decreasing its Ca2+ sensor threshold (Van Petegem 2012). We examined the effect of

BPA on RyR phosphorylation status using Western immunoblotting in isolated female rat

ventricular myocytes. BPA (1 nM) exposure increased phosphorylation of RyR at serine 2808

PKA site rapidly and transiently (Figure 3A1). The change was detected initially at 30

seconds of BPA exposure and reached peak at around 5 minutes. Compared with control, the

phospho/total-RyR level at peak was more than 2-fold (2.24 ± 1.07 fold, P < 0.05; Figure

3A2). After 15 minutes of BPA exposure, the phosphorylation level of RyR returned to

baseline. On the other hand, at the CAMKII site (serine 2814), BPA (1 nM) exposure did not

affect the phosphorylation status of RyR within a 15 minute time frame (Figure 3B1 and

3B2).

PKA’s role in mediating the rapid increase of RyR phosphorylation by BPA was

examined using PKI and H89, which are two unrelated PKA inhibitors. As shown in Figure

4A, RyR phosphorylation increase at PKA site elicited under BPA exposure was abolished by

both PKI and H89 treatment (measured at 5 minutes). By contrast, when we used AIP and

Figure 3. BPA’s rapid impact on RyR in isolated female rat ventricular myocytes. A1 and A2, Immunoblot and quantification of RyR phosphorylation status at serine 2808 PKA site under control and under exposure to BPA (1 nM). Cont., control. n = 5 hearts. B1 and B2,

Immunoblot and quantification of RyR phosphorylation status at serine 2814 CAMKII site under control and under exposure to BPA (1 nM). n = 4 hearts. All band values were normalized to control bands. *, P < 0.05 vs control; N.S., not significant.

Figure 4. BPA’s rapid impact on myocyte RyR involved cAMP/PKA signaling. A,

Immunoblot and quantification of RyR phosphorylation status at serine 2808 PKA site under control, BPA, BPA plus PKI, and BPA plus H89 treatment (n = 3 hearts). PKI: 10-6 M. H89:

10-6 M. B, Immunoblot and quantification of RyR phosphorylation status at serine 2808 PKA site under control, BPA, BPA plus AIP, and BPA plus KN93 treatment (n = 3 hearts). AIP:

10-6 M. KN93: 10-6 M. C, Intracellular cAMP levels in isolated female rat ventricular

myocytes measured by ELISA under control and under BPA, isoproterenol, and BPA plus

PHTPP treatment for 5 minutes (n = 3 hearts). Isoproterenol, Iso: 10-7 M. PHTPP: 5 x 10-5 M.

Values were expressed as fold changes to control. *, P < 0.05 vs control.

KN93, which are two unrelated CAMKII inhibitors, the increase of RyR phosphorylation at

serine 2808 under BPA exposure was not blocked (Figure 4B). These results demonstrated

that the phosphorylation level of RyR could be altered by rapid BPA exposure, which was

mediated mostly by PKA but not CAMKII.

In order to further investigate the BPA-activated PKA signaling pathway, the

intracellular cAMP level in isolated female rat ventricular myocytes was measured under

BPA exposure. PKA activation requires the production of intracellular cAMP, which is

converted from ATP by adenylyl cyclase (AC). The intracellular cAMP level was markedly

increased by more than 5-fold in the myocytes under BPA (1 nM) exposure for 5 minutes

compared with control myocytes (5.42 ± 0.34 fold, P < 0.05; Figure 4C), which was

comparable to the effect induced by -adrenergic receptor stimulation using isoproterenol

(10-7 M, for 5 minutes). Consistent with what our lab reported previously, that ER played

the dominant role in mediating the rapid effects of BPA in female myocytes (Belcher, Chen

et al. 2012), cAMP production increase elicited by BPA exposure was fully abolished by

PHTPP, an ER selective blocker (Belcher, Chen et al. 2012).

1.4. CAMKII signaling pathway in BPA’s rapid effects

In SR Ca2+ reuptake, PLN is the central regulator of SERCA function. PLN can be

phosphorylated by PKA at serine 16 and by CAMKII at threonine 17. PLN’s phosphorylation

releases the inhibitory effect on SERCA, resulting in an increase in SR Ca2+ reuptake

(Kranias and Hajjar 2012). Different from RyR’s phosphorylation pattern induced by BPA,

the phosphorylation status of PLN at the PKA site was not changed under exposure to 1 nM

BPA (Figure 5A1 and 5A2). However, the phosphorylation of PLN at threonine 17 CAMKII

Figure 5. BPA’s rapid impact on PLN in isolated female rat ventricular myocytes. A1 and A2, Immunoblot and quantification of PLN phosphorylation status at serine 16 PKA site under control and under exposure to BPA (1 nM). Cont., control. n = 3 hearts. B1 and B2,

Immunoblot and quantification of PLN phosphorylation status at threonine 17 CAMKII site under control and under exposure to BPA (1 nM) for indicated time. n = 4 hearts. All band values were normalized to control bands. *, P < 0.05 vs control; N.S., not significant.

site was increased rapidly under BPA exposure in 30 seconds and reached the peak at around

5 minutes (Figure 5B1). Average peak phospho/total-PLN level at CAMKII site was 2.22 ±

1.03 fold of control (P < 0.05; Figure 5B2). PLN phosphorylation increase was transient, and returned to the control level in 15 minutes of BPA exposure. We examined AIP and KN93, which are two unrelated CAMKII inhibitors, and found that both of them completely abolished PLN phosphorylation increase at CAMKII site elicited by BPA exposure (Figure

6A). PKI and H89, which are two PKA inhibitors, did not affect the phosphorylation of PLN under BPA exposure at CAMKII site (Figure 6B). These results indicated that BPA could rapidly increase PLN phosphorylation levels via CAMKII but not PKA.

Phospholipase C (PLC) and inositol trisphosphate receptor (IP3R)’s roles in

BPA-induced PLN phosphorylation were investigated. Using U73122 to block PLC or

xestospongin C to block IP3R (Figure 7A and 7B), the increase in CAMKII phosphorylation

of PLN was fully abolished under BPA exposure. These results indicated that the

BPA-induced CAMKII signaling pathway likely involves activation of PLC, production of

2+ 2+ IP3, and IP3R-mediated Ca release, which likely came from endoplasmic reticulum Ca .

We have also seen that blockade of PLC using U73122 did not affect RyR phosphorylation at

PKA site under BPA exposure (Figure 7C), which suggested that PLC signaling might not cross-activate cAMP/PKA signaling under BPA treatment in isolated myocytes.

1.5. Roles of estrogen receptors and ERK1/2 in BPA’s rapid effects

Earlier work in our lab has demonstrated that BPA’s rapid effects on female rat ventricular myocytes including arrhythmogenesis and alteration of myocyte mechanics were mostly mediated by estrogen receptor (ER) -signaling (Belcher, Chen et al. 2012). In this

Figure 6. BPA’s rapid impact on myocyte PLN involved CAMKII signaling. A,

Immunoblot and quantification of PLN phosphorylation status at threonine 17 CAMKII site

-6

under control, BPA, BPA plus AIP, and BPA plus KN93 treatment (n = 3 hearts). AIP: 10 M.

KN93: 10-6 M. B, Immunoblot and quantification of PLN phosphorylation status at threonine

17 CAMKII site under control, BPA, BPA plus PKI, and BPA plus H89 treatment (n = 3

hearts). PKI: 10-6 M. H89: 10-6 M. *, P < 0.05 vs control.

Figure 7. Role of phospholipase C/inositol 1,4,5-trisphosphate receptor in the rapid

effects of BPA on female rat ventricular myocytes. A, Immunoblot and quantification of

PLN phosphorylation status at threonine 17 CAMKII site under control, BPA, BPA plus

U73122 treatment. U73122: 10-6 M. B, Immunoblot and quantification of PLN

phosphorylation status at threonine 17 CAMKII site under control, BPA, BPA plus

xestospongin C treatment. Xesto: Xestospongin C, 5 x 10-6 M. C, Immunoblot and

quantification of RyR phosphorylation status at serine 2808 PKA site under control, BPA,

and BPA plus U73122 treatment. U73122: 10-6 M. BPA = 1 nM.

study, ER and ER’s roles in mediating BPA’s rapid effects on increasing RyR and PLN

phosphorylation levels were examined (Figure 8A and 8B). ER blocker, PHTPP, fully

blocked BPA-induced PLN phosphorylation increase at the CAMKII site and RyR

phosphorylation increase at the PKA site, but ER blocker MPP showed no blocking effects.

It was reported that in a variety of cells types, ERK mitogen-activated protein kinase

(MAPK) signaling played a critical role in mediating BPA rapid signaling (Wetherill,

Akingbemi et al. 2007). ERK1/2 phosphorylation under BPA exposure in female rat

ventricular myocytes was assessed. There was no detectable change in the level of ERK1/2

phosphorylation within 15 minutes (Figure 8C), indicating that MAPK pathway may not involve in the rapid actions of BPA in female rat ventricular myocytes.

1.6. Functional endpoints analysis: roles of PKA and CAMKII

1.6.1. SR Ca2+ release/leak

It was demonstrated that spontaneous SR Ca2+ release, or SR Ca2+ leak, played a critical role in BPA’s pro-arrhythmic effects in the female rat myocytes (Yan, Chen et al. 2011).

Using confocal microscopy, SR Ca2+ leak in quiescent myocytes under BPA and kinase blockers treatment was examined. Inhibition of PKA by PKI or H89 fully blocked the

increase in SR Ca2+ leak (measured as Ca2+ spark frequency) under BPA treatment, which

was consistent with previous RyR western immunoblotting results (Figure 9A and 9B). By contrast, inhibition of CAMKII by AIP or KN93 did not show any effects on Ca2+ spark with

BPA treatment (Figure 9D and 9E). No changes in peak amplitude (Figure 9C and 9F) or

spatial/temporal properties (data not shown) of Ca2+ spark was observed for all the treatments.

These results indicated that the increase in SR Ca2+ release under BPA exposure was mostly

Figure 8. Role of ERs and ERK1/2 in the rapid effects of BPA on female rat ventricular

myocytes. A and B, Immunoblots and quantifications of PLN phosphorylation status at

threonine 17 CAMKII site and RyR phosphorylation status at serine 2808 PKA site under

control, BPA, BPA plus MPP, and BPA plus PHTPP treatment. MPP: 10-6 M. PHTPP: 5 x

10-6 M. C, Immunoblot and quantification of ERK1/2 phosphorylation status at p42/ p44

under control and under exposure to BPA for indicated time. BPA = 1 nM.

Figure 9. SR Ca2+ leak in female rat ventricular myocytes under BPA exposure was

mediated by PKA but not CAMKII. A and D, Effects of PKA inhibitor PKI and CAMKII

blocker AIP on Ca2+ spark (indicated by the red arrow) induced by BPA treatment in

quiescent myocytes. B and E, Average data on the frequency of Ca2+ spark. C and F, Average

data on the amplitude of Ca2+ spark (n = 6 hearts). For B and C, data were from 128 sparks in

10 myocytes of 4 hearts. For E and F, data were from 206 sparks in 22 myocytes of 6 hearts.

BPA = 1 nM. PKI, H89, AIP, and KN93 = 10-6 M. *, P < 0.05 vs control; N.S., not significant.

the result of increased RyR phosphorylation via PKA.

1.6.2. Arrhythmogenic-triggered activity

Spontaneous Ca2+ transients in isolated female rat ventricular myocytes following

electrical pacing (Ca2+ after-transients) were measured to evaluate myocyte triggered

activities. BPA rapidly increased the percentage of myocytes showing triggered activities

(Figure 10A and 10B, control vs. BPA: 6% vs. 29%, P < 0.05). PKA inhibitors, PKI and H89, and CAMKII blockers, AIP and KN93, all abolished the pro-arrhythmic effects of BPA treatment (Figure 10A and 10B). These results indicated that PKA and CAMKII are both indispensible to BPA’s arrhythmogenic action in female rat ventricular myocytes.

1.6.3. Myocyte contractility

In order to gauge BPA’s impact on Ca2+ handling in the myocytes, contractility of the myocytes was previously measured to serve as an index. Here, BPA rapidly increased the contractility of isolated female rat ventricular myocytes. Fractional shortening of myocytes

was increased from 8.6 % to 14.3% under BPA exposure (P < 0.05), which was fully blocked

by PKA inhibitors PKI and H89. Interestingly, BPA’s effect on enhancing myocyte

contractility was not affected by CAMKII blockers AIP or KN93 (Figure 11A and 11B).

These results indicated that BPA’s stimulatory effect on female rat myocyte contractility was

primarily mediated by PKA signaling.

Discussion 1

Previously our lab showed that BPA, an environmental estrogenic endocrine disrupting

chemical, rapidly promoted cardiac arrhythmias in female adult rat hearts via alteration of

Ca2+ handling (Yan, Chen et al. 2011). This dissertation elucidated the underlying mechanism

Figure 10. PKA and CAMKII’s roles in mediating BPA’s action on triggered activity. A,

Example confocal microscopic line scan images of Ca2+ transients induced by electrical

pacing in myocytes under control, BPA, BPA plus PKI, and BPA plus AIP. Spontaneous

Ca2+ after-transient after pacing (ie, triggered activity) were indicated by red arrows. B, Data

on percentages of myocytes showing triggered activities under different conditions. n = 33 –

49 myocytes in 3 hearts. BPA = 1 nM. PKI, H89, AIP, and KN93 = 10-6 M. *, P < 0.05 vs

control.

Figure 11. PKA and CAMKII’s roles in mediating BPA’s action on myocyte

contractility. A, Example contraction traces of isolated female rat ventricular myocytes

under control, BPA, BPA plus PKI, BPA plus AIP, and BPA plus KN93. B, Average data on

myocyte fractional shortening under control, BPA, BPA plus PKI, BPA plus H89, BPA plus

AIP, and BPA plus KN93. n = 21 – 55 myocytes in 4 hearts. BPA = 1 nM. PKI, H89, AIP,

and KN93 = 10-6 M. *, P < 0.05 vs control.

of the cardiac-specific rapid actions of BPA, particularly the signaling pathways involved.

PKA and CAMKII signaling pathways play major roles in mediating BPA’s rapid impact on

Ca2+ handling and arrhythmogenesis in female rat ventricular myocytes (Figure 12). With

BPA exposure to the myocytes, cAMP production is rapidly increased with adenylyl cyclase

activation, which leads to activation of PKA and phosphorylation of RyR; increased RyR

phosphorylation enhances RyR opening probabilities and hence SR Ca2+ leak. On the other

hand, BPA exposure also rapidly activates Phospholipase C and increases inositol

2+ 1,4,5-trisphosphate (IP3) production. IP3R-mediated Ca release, which likely comes from

the Ca2+ stored in endoplasmic reticulum, activates CAMKII to increase PLN

phosphorylation. Inhibition of PLN on SERCA is released by PLN phosphorylation, and

hence SR Ca2+ reuptake is increased. These two signaling pathways both contribute to

myocyte triggered activities induced by BPA, and increase arrhythmogenesis to the female

heart (Figure 12). Both signaling pathways are mediated by ER in a rapid but transient

manner. Most likely, these two signalings are localized and impact only on their specific

target proteins. ERK1/2, though commonly implicated in the rapid signaling of BPA (Belcher,

Le et al. 2005), does not participate in the rapid arrhythmogenic effects of BPA in the heart.

It was shown that cAMP level was rapidly increased in female rat ventricular myocytes

under BPA exposure. The increase level was comparable to full -adrenergic activation by

isoproterenol. Activation of cAMP/PKA signaling by BPA has been previously described in

different types of tissues, including neurons, placenta and various cancer cell lines (Bouskine,

Nebout et al. 2008; Bouskine, Nebout et al. 2009; Huang, Tan et al. 2012). PKA activation,

which is the downstream effect of cAMP production, rapidly increased RyR phosphorylation

Figure 12. Illustration of BPA’s rapid signaling cascade in female rat ventricular myocytes.

but did not affect PLN phosphorylation. Thus, BPA acts distinctly from -adrenergic agonists

which lead to PKA’s phosphorylation of both RyR and PLN. RyR phosphorylation is

considered critical for regulating excitation-contraction coupling in cardiac myocytes and

also plays the key role in cardiac arrhythmogenesis (Bers 2002). In heart failure, it was

demonstrated that RyR becomes hyper-phosphorylated by PKA due to RyR macromolecular

complex remodeling and loss of phosphatase activities of PP1 and PP2A (Marx, Reiken et al.

2000). Hyper-phosphorylated RyR channel affects its normal function resulting in Ca2+ leak

from SR into cytosol, and induces development of life-threatening cardiac ventricular

arrhythmias (Marx, Reiken et al. 2000). These observations under pathological conditions are

supportive of the idea that PKA phosphorylation of RyR induced by BPA may play a

detrimental role in arrhythmogenesis. Indeed, either PKA inhibition (Figure 10) or RyR

opening blockade (Yan, Chen et al. 2011) abolished development of triggered activities under

BPA exposure.

The fact that BPA can modulate intracellular Ca2+ has been reported in various types of

cells including hippocampal neurons, renal tubular cells, islet of Langerhans and prostate

cancer cells (Alonso-Magdalena, Laribi et al. 2005; Tanabe, Kimoto et al. 2006; Lee, Suk et al. 2008; Kuo, Huang et al. 2011; Soriano, Alonso-Magdalena et al. 2012; Derouiche,

Warnier et al. 2013). Though intracellular Ca2+ elevation and activation of a Ca2+-responding

kinase CAMKII are most likely linked, our study is the first one to directly show the

importance of the CAMKII signaling pathway in mediating BPA’s rapid cardiac effects. In

the heart, being a highly coordinated process, Ca2+ handling is fine-tuned by several Ca2+

dependent mechanisms, one of which is CAMKII. CAMKII is activated under -adrenergic

stimulation with elevated intracellular Ca2+ levels, and it phosphorylates downstream target

proteins, including several Ca2+ handling proteins, to regulate the function of myocytes (Bers

2008). When PLN is phosphorylated by CAMKII, it de-inhibits SERCA function so the Ca2+

reuptake process is enhanced, which enables faster relaxation and refilling of the heart under

elevated heart rate conditions (Maier and Bers 2007). Under pathological conditions,

activation of CAMKII favors dysfunction and electrical instability of the heart. In animal

experiments, several forms of arrhythmias have been linked with activation of CAMKII such

as atrial fibrillation, sinus node dysfunction, ventricular tachycardias and inherited

tachycardias (Swaminathan, Purohit et al. 2012). A CAMKII expression increase, in disease conditions such as heart failure, could disturb homeostasis of cytosolic Ca2+ via

hyper-phosphorylating several Ca2+ handling proteins, which leads to development of triggered activities and potentially arrhythmias in the heart (Rokita and Anderson 2012). In this dissertation, CAMKII activation was essential to triggered activities development in myocytes under BPA exposure, which was consistent with CAMKII’s role in arrhythmogenesis under disease conditions. However, it was not shown that CAMKII only phosphorylated PLN in the heart, and exactly how it influences cardiac function is still not known. The consequence of this unique impact of BPA on myocyte Ca2+ handling remains to

be elucidated.

Notably, CAMKII’s activation in myocytes under BPA exposure was mediated by PLC,

which was distinct from the conditions commonly described physiologically and

pathophysiologically. CAMKII activation is a secondary process to the activation of PKA

under -adrenergic stimulation. Elevated intracellular Ca2+ resulting from increased SR Ca2+

release and L-type channel Ca2+ influx are the main sources for CAMKII’s Ca2+ dependent

activation (Grimm and Brown 2010). However, BPA activated CAMKII independently from

the cAMP/PKA pathway. Blocking PKA using PKI abolished PKA phosphorylation on RyR,

but had no effects on CAMKII phosphorylation of PLN. Blockers of PLC or IP3R fully

abolished activation of CAMKII as measured by PLN phosphorylation, indicating that

distinct from -adrenergic stimulation, the Ca2+ source for CAMKII activation in myocytes

under BPA exposure is from endoplasmic reticulum via IP3R, induced by activation of PLC

and production of IP3.

Also, the BPA-induced cAMP/PKA pathway is not cross-activated by the PLC pathway

in cardiac myocytes. It is previously shown that PKA is a downstream effector of AC/cAMP

signaling activated by Gs-coupled membrane bound ER (Levin 2008), and this appears to be

the case on myocytes in the present study. On the other hand, PKA can also be activated by

Gq-coupled membrane bound ER in hypothalamic neurons via PLC-diacylglycerol- protein

kinase C (PKC) activation. PKC phosphorylates and activates AC, increasing intracellular cAMP production (Qiu, Bosch et al. 2003; Kelly and Ronnekleiv 2012). However, in cardiac

myocytes, such PLC-PKC-AC signaling seems not to play a major role in BPA-induced PKA

activation. The PLC blocker, while abolishing activation of CAMKII in myocytes, had no

effects on PKA activation under BPA exposure (Figure 7).

The fact that both cAMP/PKA pathway and PLC/IP3R/CAMKII pathway can be

activated by BPA suggests that membrane-associated ERs are likely to couple with more than

one type of G-proteins. It was previously reported that activation of PACAP (pituitary

adenylate cyclase-activating polypeptide type 1) receptors resulted in both cAMP elevation

and IP3 production, in a comparable pattern with BPA’s myocyte signalings. It was also

shown in this study that different ligands, peptides PACAP1-27 and PACAP1-38, have different efficacies in terms of increasing the production of cAMP or IP3 (Spengler, Waeber

et al. 1993). This type of agonist activation, not in a uniform but a “biased” mechanism,

toward some instead of all the signaling pathways is called biased agonism (Kenakin 2009).

In our preliminary experiments using endogenous estrogen 17β-estradiol (E2),

phosphorylations of RyR and PLN were both increased, showing similar levels with BPA

exposure to the myocytes. It indicates that E2 and BPA may have the same pattern to activate

cAMP/PKA and PLC/IP3/CAMKII signaling pathways, and no biased agonism was shown in

examination of these two ligands.

It is also interesting that although RyR and PLN both have phosphorylation sites for

PKA and CAMKII, in cardiac myocytes with BPA exposure PKA and CAMKII phosphorylate RyR and PLN preferentially. Such specific targeting actions are likely due to a localized/compartmentalized signaling mechanism. Both kinases indeed have been reported to show target-specific phosphorylating actions in cardiac myocytes (McConnachie,

Langeberg et al. 2006; Maier and Bers 2007). PKA can control cAMP signal transduction both temporally and spatially through interaction with A-kinase anchoring proteins (AKAPs), which are intracellular scaffolding proteins (McConnachie, Langeberg et al. 2006). RyR binds to AKAP complex and is phosphorylated by PKA on the nucleus envelope of cardiac myocytes (Kapiloff, Jackson et al. 2001). CAMKII can also co-localize with target proteins and phosphatases to modulate activation of transcription factors and regulate gene expression

in cardiac myocytes (Maier and Bers 2007). BPA likely activates such a localized signaling

cascade to impact myocyte Ca2+ handling process.

Our data shows that ERK1/2 phosphorylation was not altered in female myocytes within

15 minutes of BPA exposure. ERK1/2 has previously been shown to participate in BPA’s

rapid signaling network in a variety of tissues and cell lines including placenta, cerebellum

granule cells, immune cells, breast cancer cells and human testicular cancer cells (Belcher, Le

et al. 2005; Wetherill, Akingbemi et al. 2007; Bouskine, Nebout et al. 2008; Huang, Tan et al.

2012; Ptak and Gregoraszczuk 2012), but not to be involved in pituitary cells or human

seminoma cells (Wetherill, Akingbemi et al. 2007; Bouskine, Nebout et al. 2009). ERK1/2

activation is associated with proliferation, migration and invasion of cancer cells (Bouskine,

Nebout et al. 2009; Ptak and Gregoraszczuk 2012). While ERK1/2 phosphorylation status

under longer-term BPA exposure was not examined, the lack of any detectable change in 15

minutes of BPA exposure suggests that ERK1/2 may not play a role in BPA’s rapid impact

on myocyte Ca2+ handling and arrhythmogenesis, which occurs rapidly (within minutes) after

BPA exposure. These results further support the tissue/cell-type specificity of BPA’s actions.

In summary, this dissertation elucidated BPA’s rapid signaling cascade in cardiac myocytes, and provided insights on molecular mechanisms underlying BPA’s rapid impact on Ca2+ handling and arrhythmogenesis in female rat cardiac myocytes. The cAMP/PKA

2+ pathway and the PLC/ IP3/Ca /CAMKII pathway are activated by BPA in myocytes in a rapid manner. They impact the phosphorylation status of RyR and PLN, which are two key

Ca2+ handling proteins in cardiac myocytes. These actions of BPA underlie its stimulatory

effect on triggered activities and arrhythmogenesis in the female heart. Results of this study

contribute to the assessment of potential cardiac toxicity of BPA and its consequences.

Result 2. Bisphenol S, a manufacturing substitute for bisphenol A, shows rapid pro-arrhythmic effects in female rat hearts

2.1. Background and rationale

In recent years, the potential adverse impact of BPA on human health called for actions

to reduce its use in consumer products. Both European Union and US Food and Drug

Administration have banned BPA’s usage in plastic bottles and drinking cups for infants and

children. France has recently issued a ban on BPA’s usage in manufacturing and sale of food

packaging starting in 2015 (EuropeanCommission 2011; Legifrance.gouv.fr. 2012; USFDA

2012).

Bisphenol S (BPS, 4,4’-Sulfonyldiphenol, CAS# 80-09-1), composed of two phenol

groups and a sulfonyl group, is structurally similar to BPA (Figure 13). BPS has been used as

a substitute plasticizing agent for BPA in many “BPA-free” labeled consumer goods. BPS is

also used in manufacturing of thermal papers including envelopes, receipts, and boarding

passes (Liao, Liu et al. 2012). Though claimed as having better heat-stability and

sunlight-resistance than BPA, BPS still leaches from food/beverage containers and cans under

normal use conditions (Vinas, Campillo et al. 2010). Human exposure to BPS has recently

been described to be wide spread. In 81% of urine samples from sample populations in US

and seven Asian countries, BPS was detected with a mean concentration of 0.65 ng/mL (2.6

nM) (Liao, Liu et al. 2012). It has been reported that BPS has estrogenic activities, similar to

BPA (Hashimoto, Moriguchi et al. 2001; Grignard, Lapenna et al. 2012). Though BPA’s

health impact has been quite extensively investigated, current knowledge on biological

actions of BPS and its potential health impact is very limited.

Figure 13. Structures of bisphenol S and bisphenol A (BPS and BPA).

Previously our lab demonstrated that low-dose BPA rapidly promoted development of arrhythmogenic-triggered activities in female rat ventricular myocytes (Yan, Chen et al.

2011). Under stress or ischemia conditions, BPA increased the risk for cardiac arrhythmias development in female rat hearts (Yan, Chen et al. 2011; Yan, Song et al. 2013). These

results indicate that BPA has potentially adverse impacts on the cardiovascular system.

However, the actual toxicity of BPS to the heart is currently unknown. In this study, the rapid

action of low-dose BPS on rat hearts and isolated cardiac myocytes were investigated, with a

focus on its arrhythmogenic effects and the underlying cellular as well as molecular

mechanisms.

2.2. BPS promoted development of ventricular arrhythmias in female rat hearts

Surface electrocardiography (ECG) examined the rapid impact of BPS on the electrical

rhythm of ex vivo hearts from female adult rats. Under control conditions, all the hearts

showed normal sinus rhythms. BPS exposure at 10-9 M did not induce any detectable

arrhythmias, but moderately increased heart rates from 290 to 319 beats/min (P < 0.05;

Figure 14A and 14B). Under catecholamine-induced stress condition created by the

-adrenergic agonist isoproterenol (10-8 M), BPS exposure at 10-9 M markedly increased

development of premature ventricular beats (PVBs) in the female rat heart, from 0.87 events

at baseline (i.e., Iso alone) to 8.83 events with BPS plus Iso in 20 minutes of recordings

(Figure 15A and 15B, P < 0.05). This result was comparable to that of BPA; under 10-9 M

BPA plus Iso, frequency of PVBs was 9.00 events in 20 minutes (Figure 15B). BPS was also

found to induce episodes of non-sustained ventricular tachycardia (VT) in the presence of Iso

(Figure 15A, 1 out of 6 hearts). Under Iso alone, no hearts developed non-sustained VT.

Figure 14. Acute BPS exposure increased heart rate in female rat hearts. A, Left panel, example surface ECG recording traces from female adult rat hearts under control, or under

10-9 M BPS exposure. Right panel, overlay of the same traces on left panel to show heart rate

increase under 10-9 M BPS exposure. Heart rate alteration was observed within 5 minutes of

BPS exposure. B, Average data on heart rate in female rat hearts under control or under 10-9

M BPS exposure. For each group, n = 3 hearts. *: P < 0.05 vs. control.

Figure 15. Acute BPS exposure promoted ventricular arrhythmias in female rat hearts.

A, Example surface ECG traces from female adult rat hearts under 10-8 M isoproterenol (Iso)

alone or under 10-9 M BPS plus 10-8 M Iso. VT: ventricular tachycardia. Red asterisks

indicate premature ventricular beats (PVBs). B, Average data on the number of premature

ventricular beats in female rat hearts within 20 minutes of recording under the indicated conditions. n = 6 - 8 hearts. *: P < 0.05 vs. control.

2.3. BPS rapidly promoted development of arrhythmogenic-triggered activities in

female rat ventricular myocytes

The effect of BPS on the development of spontaneous excitations in isolated female rat

ventricular myocytes were examined. These aberrant spontaneous excitations in myocytes are

known as “triggered activities”, in the present study recorded as spontaneous Ca2+ transients

following repeated electrical pacing (Ca2+ after-transients) (Bers 2002). Exposure to 10-9 M

BPS for 2-7 min induced the development of triggered activities in 30% of female rat

ventricular myocytes (Figure 16A and 16B), while triggered activities were only recorded in

6.6% of female rat ventricular myocytes under control condition (P < 0.05). The effect of 10-9

M BPS on promoting triggered activity in myocytes was comparable to that of 10-9 M BPA

(Figure 16B). Different from the robust stimulatory effect observed at 10-9 M, BPS at 10-12 M or 10-6 M did not increase the percentage of myocytes with triggered activities, resulting in an

inverted-U shaped dose response (Figure 16B).

2.4. BPS rapidly affected Ca2+ handling in female rat ventricular myocytes

The Ca2+ handling process is fundamental to cardiac myocyte physiology; abnormalities

in Ca2+ handling are important contributors to cardiac arrhythmogenesis (Bers 2002).

Previously, BPA’s pro-arrhythmic action on the heart was shown to be mediated by alteration

of myocyte Ca2+ handling (Yan, Chen et al. 2011). In the present study, the rapid effects of

BPS (10-9 M) on Ca2+ handling in female rat ventricular myocytes were investigated. BPS

2+ rapidly increased field-stimulated Ca transient amplitude from 1.88 to 4.80 (F/F0 ratio)

(Figure 17A and 17B). The time constant (tau) was significantly decreased under BPS

exposure from 595.0 ms to 386.3 ms (Figure 17B), suggesting an increase in Ca2+ reuptake

Figure 16. Acute exposure to BPS induced triggered activities in female rat ventricular

myocytes. A, Example confocal microscopic line scan images showing Ca2+ transients in

female rat ventricular myocytes elicited by electrical pacing under control and under 10-9 M

BPS exposure. Red arrows: spontaneous Ca2+ after transients (i.e., triggered activities). B,

Data on percentage of female rat ventricular myocytes with triggered activities under

indicated conditions. n = 30 myocytes of 4 hearts for each group. *: P < 0.05 vs. control.

Figure 17. BPS rapidly altered Ca2+ transient in female rat ventricular myocytes. A,

Example Ca2+ transient traces in female rat ventricular myocytes induced by electrical

stimulation under control or under 10-9 M BPS exposure. B, Average data on the amplitude

(CaT) and decay time constant (tau) of Ca2+ transient in female rat ventricular myocytes

under control or under 10-9 M BPS exposure. n = 6 - 8 myocytes of 3 hearts. *: P < 0.05 vs.

control.

rate. It is known that increased SR Ca2+ release in diastole, or SR Ca2+ leak, plays an

important role in BPA’s cardiac arrhythmogenesis (Gao, Liang et al. 2013). SR Ca2+ release

was examined in quiescent female rat ventricular myocytes by measuring Ca2+ spark. 10-9 M

BPS exposure rapidly and significantly increased the frequency of Ca2+ spark from 1.36 to

2.18 sparks per second per 100 μm (Figure 18A and 18B); while it did not affect the peak

amplitude of Ca2+ spark (Figure 18C). PHTPP, a selective ERβ blocker fully abolished the

effects of BPS on Ca2+ spark (Figure 18).

The contractility of myocytes manifests the process of Ca2+ cycling; hence, in the present

study it was used as a global index to further examine BPS action on myocytes. BPS increased fractional shortening in female rat ventricular myocytes (Figure 19). The enhancing

effects were dose-dependent with an inverted-U shape curve. The efficacy of BPS was

comparable to BPA, and they both showed the most efficacious response at 10-9 M and 10-8

M (Figure 19A). BPS plus BPA (both at 10-9 M) did not increase the effect, suggesting they

did not have antagonistic nor synergistic actions (Figure 19B). PHTPP, the selective ERβ

blocker completely abolished BPS actions on myocyte contractility, but MPP, the selective

ERα blocker did not show any blocking effects (Figure 19B).

2.5. BPS rapidly affected Ca2+ handling proteins in female rat ventricular myocytes

Ryanodine receptor (RyR) and phospholamban (PLN) are two important proteins to

regulate key steps in myocyte Ca2+ handling, including SR Ca2+ release and reuptake of Ca2+.

Their activities can be modulated via protein kinase phosphorylation (Kranias and Hajjar

2012; Van Petegem 2012). The effects of BPS on RyR and PLN phosphorylation in female rat

ventricular myocytes were examined by western immunoblot (Figure 20). Similar with

Figure 18. BPS rapidly altered Ca2+ spark in female rat ventricular myocytes. A,

Example confocal microscopic line scan images of Ca2+ spark (indicated by the red arrow) in

female rat ventricular myocytes under control, 10-9 M BPS or 10-9 M BPS plus 5 x 10-6 M

PHTPP treatment. (B) and (C) are averages data on the frequency and amplitude of Ca2+

spark under control, 10-9 M BPS or 10-9 M BPS plus 5 x 10-6 M PHTPP treatment. n = 6 - 7

myocytes of 3 hearts. *: P < 0.05 vs. control. N.S., not significant.

Figure 19. BPS rapidly altered contractility of female rat ventricular myocytes. A, Dose

response curve of BPS effects on the myocyte contractility as measured by fractional

shortening in female rat ventricular myocytes. The effect of 10-9 M BPA on myocyte

fractional shortening was also shown. n = 25 - 26 myocytes of 4 hearts. B, Fractional

shortening of myocytes under indicated treatments. PHTPP: 5 x 10-6 M. MPP: 10-6 M. n = 11

- 12 myocytes of 3 hearts. *: P < 0.05 vs. control.

what our lab observed with BPA (Gao, Liang et al. 2013), 10-9 M BPS exposure rapidly and transiently increased RyR phosphorylation level at serine 2808, which is a protein kinase A

(PKA) site (Figure 20A), and PLN at threonine 17, which is a Ca2+/CaM-dependent protein

kinase II (CAMKII) site (Figure 20C). The changes in phosphorylation levels were detected

at 30s upon BPS exposure and reached peak at around 5 minutes. BPS did not alter RyR

phosphorylation at the CAMKII site (serine 2814) nor PLN at the PKA site (serine 16)

(Figure 20B and 20D). The ERβ blocker, PHTPP, but not the ERα blocker MPP, completely abolished BPS effects on RyR and PLN phosphorylation (Figure 21).

2.6. BPS gender specificity in cardiac actions

The effects of BPS in male adult rats were investigated. Different from the arrhythmogenic effects observed from female rat hearts, BPS did not have similar effects on male rat hearts. At the whole heart level, the frequency of premature ventricular beats under

10-9 M BPS plus Iso was the same with that under Iso alone in males (Figure 22A and 22B,

P > 0.3). Heart rate was not altered by 10-9 M BPS exposure (Figure 22C). At the myocyte

level, BPS did not affect the development of triggered activities in male rat ventricular

myocytes (Figure 22D), nor alter Ca2+ spark properties (Figure 23A) or Ca2+ transient

kinetics (Figure 23B). The phosphorylation statuses of RyR and PLN at either PKA or

CAMKII sites were not affected by rapid BPS exposure in male rat ventricular myocytes

(Figure 24).

The mechanism underlying male rat myocytes’ lack of response to rapid BPS exposure

was assessed with triggered activity development and RyR phosphorylation at serine 2808

PKA site in isolated male rat myocytes as endpoints. While BPS did not impact the

Figure 20. BPS rapidly increased RyR and PLN phosphorylation in female rat myocytes.

A and B, Immunoblots and quantifications of RyR phosphorylation at serine 2808 PKA site and serine 2814 CAMKII site in female rat ventricular myocytes under control and under 10-9

M BPS exposure for indicated time points. Cont., control. C and D, Immunoblots and quantifications of PLN phosphorylation at threonine 17 CAMKII site and serine 16 PKA site in female rat ventricular myocytes under control and under 10-9 M BPS exposure for

indicated time points. n = 3 hearts. *: P < 0.05 vs. control, N.S., not significant.

Figure 21. BPS increased RyR and PLN phosphorylation through ERβ. A, Immunoblot

and quantification of RyR phosphorylation at serine 2808 PKA site in female rat ventricular

myocytes under control, 10-9 M BPS, 10-9 M BPS plus 10-6 M MPP, 10-9 M BPS plus 5 x 10-6

M PHTPP treatment. B, Immunoblot and quantification of PLN phosphorylation at threonine

17 CAMKII site in female rat ventricular myocytes under control, 10-9 M BPS, 10-9 M BPS

plus 10-6 M MPP, 10-9 M BPS plus 5 x 10-6 M PHTPP treatment. n = 3 hearts. *: P < 0.05 vs. control.

Figure 22. BPS gender specificity in cardiac arrhythmogenesis in male rat hearts. A,

Example surface ECG recordings in male adult rat hearts under the treatment of 10-9 M BPS

plus 10-8 M isoproterenol. Premature ventricular beat (PVB) is indicated by a red asterisk. B,

Average data on the number of PVBs in 20 minutes of recordings in male adult rat hearts

under indicated conditions. n = 5 - 6 hearts. C, Average data on male rat heart rate (beats per

min) under control or under 10-9 M BPS exposure. n = 5 hearts. D, Data on percentage of

male adult rat ventricular myocytes showing triggered activities under indicated conditions. n

= 42 - 43 myocytes of 4 hearts. N.S., not significant.

Figure 23. BPS gender specificity in cardiac Ca2+ handling in male rat ventricular

myocytes. A, Left, example confocal microscopic line scan images of Ca2+ spark in male

adult rat ventricular myocytes under control and under 10-9 M BPS exposure. Right, average

data on the frequency and peak amplitude of Ca2+ spark under control and under 10-9 M BPS

exposure. n = 10 myocytes of 3 hearts. B, Average data on the decay time constant of Ca2+ transient under control and under 10-9 M BPS exposure. n = 13 myocytes of 3 hearts. N.S.,

not significant.

Figure 24. BPS gender specificity in phosphorylation status of RyR and PLN. A and B,

Immunoblots and quantification of RyR phosphorylation at serine 2808 PKA site and serine

2814 CAMKII site from male adult rat ventricular myocytes under control and under 10-9 M

BPS exposure for indicated time points. Cont., control. C and D, Immunoblots and

quantification of PLN phosphorylation at threonine 17 CAMKII site and serine 16 PKA site

from male adult rat ventricular myocytes under control and under 10-9 M BPS exposure for

indicated time points.

development of triggered activities or RyR phosphorylation in male adult rat ventricular

myocytes, ER activation with a selective agonist DPN markedly enhanced the incidence of

triggered activities (Figure 25A) and level of RyR phosphorylation at serine 2808 (Figure

25B). Similar stimulatory effects were observed when MPP, a selective ERα blocker, was

used together with BPS to treat the myocytes. MPP alone had no detectable effects (Figure

25A and 25B).

Discussion 2

BPS, another member of the bisphenol family having a similar chemical structure to BPA,

is becoming a BPA alternative chemical in the manufacturing of consumer products due to

recognition of BPA’s potential adverse impact on human health. It is shown by a recent study

that BPS is currently a common environmental substance (Liao and Kannan 2013). However,

current knowledge of BPS and other BPA substitutes’ biological effects and potential

toxicological impact is lacking.

This dissertation shows that BPS at low-dose has rapid impacts on female rat hearts at the organ, cellular, and protein levels. These effects were specific to females but not males, and strikingly paralleled BPA’s cardiac pro-arrhythmic effects at equivalent doses (Yan, Chen et al. 2011). These results demonstrate that at a low-dose relevant to environmental exposure level, BPS could have potential adverse cardiac effects on females, and also suggest that BPS as well as other BPA substitute chemicals may not be necessarily safe and free of the adverse effects identified for BPA.

Limited but increasing evidence suggests that BPS, like its analog BPA, has potential estrogenic endocrine disrupting activities. Studies have shown that BPS has genomic

Figure 25. Roles of ERα and ERβ in BPS gender specificity in male rat ventricular

myocytes. A, Data on percentage of male adult rat ventricular myocytes showing triggered

activities under indicated conditions. n = 24 myocyte of 3 hearts. B, Immunoblots and

quantification of RyR phosphorylation at serine 2808 PKA site in male adult rat ventricular

myocytes under various treatments. Cont., control. BPS: 10-9 M, DPN: 10-7 M, MPP: 5 x 10-6

M. *: P < 0.05 vs. control.

estrogenic activities in various cell lines at supra-physiological doses from μM to mM

(Hashimoto, Moriguchi et al. 2001; Kuruto-Niwa, Nozawa et al. 2005; Grignard, Lapenna et

al. 2012). Recent studies demonstrated that BPS exposure adversely affected zebrafish

reproduction and progeny generation at nM concentrations (Ji, Hong et al. 2013), and

significantly altered production of progesterone, testosterone and cortisol in human adrenal

cortico-carcinoma cells (Rosenmai, Dybdahl et al. 2014). In addition, acute exposure to BPS

at low-dose was shown to activate ERK signaling in a pituitary cell line (Vinas and Watson

2013). In addition, BPS exposure at 20 and 500 mg/kg dose for three consecutive days

increased female rat uterine weight (Yamasaki, Noda et al. 2004).

This dissertation reports that BPS at nM doses rapidly increased the development of

spontaneous excitations (“triggered activities”) in female adult rat ventricular myocytes.

Triggered activities are arrhythmic events on the myocyte level, and could propagate through

the myocardium under certain pathological conditions. This has been established as one of

the central cardiac arrhythmogenic mechanisms (Bers 2002). BPS exposure alone moderately

increased heart rate in female rats at the whole heart level, but did not induce any arrhythmic

events. A previous study showed that in female rat ventricular myocytes BPA exposure

increased intracellular cAMP level (Gao, Liang et al. 2013). It is known that the pacemaker current and automaticity rate of cardiac nodal cells are regulated by cAMP binding and PKA activation (Baruscotti and Difrancesco 2004). Although no investigation was performed on the impact of BPS on pacemaker cells, the mechanism underlying BPS’ increasing heart rate in female rats likely involves intracellular cAMP. BPS’ effects on rhythm of the heart were examined under the stress condition induced by catecholamine. Catecholamine increases Ca2+

influx as well as SR Ca2+ load in the myocytes, which favors SR Ca2+ overload and abnormal

SR Ca2+ release, providing a substrate for cardiac arrhythmogenesis (Bers 2002). Under such

conditions, BPS exposure induced frequent premature ventricular beats, which are one of the most common ventricular arrhythmias. These effects of 10-9 M BPS were comparable to

those of 10-9 M BPA. Our data suggest that though BPS exposure is not likely to result in

clinically relevant cardiac arrhythmias by itself under normal conditions in healthy people, it could contribute to arrhythmia development under pathological states such as increased sympathetic tone and cardiovascular diseases.

The underlying mechanism of BPS’ arrhythmogenic action in the female rat heart was explored, which involves rapid alteration of Ca2+ handling in the myocytes, similar to BPA.

Rapid BPS exposure significantly enhanced SR Ca2+ reuptake process and diastolic SR Ca2+ release (Ca2+ leak) which is a pathophysiological alteration playing important roles in cardiac arrhythmogenesis (Chelu and Wehrens 2007). Our lab has shown previously that SR Ca2+

leak suppression abolished BPA-induced triggered activities in female myocytes (Yan, Chen et al. 2011). The effects of BPS on myocyte Ca2+ handling were mediated by phosphorylation

of RyR and PLN which are two key Ca2+ handling proteins. An almost identical effect was

observed for BPA (Chapter III: Result 1). Rapid BPS exposure increased RyR

phosphorylation by PKA, but not by CAMKII; increased PLN phosphorylation by CAMKII,

but not by PKA. This effect occurred in a transient manner. Both the time course of action

and the target-specificity of BPS are remarkably similar to previously described BPA cardiac

actions (Chapter III: Result 1). Adenylyl cyclase-cAMP-PKA signaling and phospholipase

C-inositol trisphosphate receptor-CAMKII signaling contribute to BPA’s impact on

arrhythmogenesis in the myocytes (Chapter III: Result 1). A similar signaling mechanism

may also apply to the rapid cardiac actions of BPS.

In terms of BPS pharmacodynamic properties, it was shown that BPS had equal potency

(EC50) with BPA in a heterologous expression system to activate an estrogen-responding GFP

reporter gene (Kuruto-Niwa, Nozawa et al. 2005). A more recent study demonstrated that

BPS was less potent than BPA, as measured by binding to ER and ER, in a competitive

binding assay and activating ER and ER-driven luciferase reporter genes (Molina-Molina,

Amaya et al. 2013). Another study found that BPS had anti-androgen effects and that its

potency was lower than BPA (Kitamura, Suzuki et al. 2005). This dissertation shows that

BPS’ pharmacodynamic properties closely resemble BPA as measured by the rapid action on

cardiac myocytes. When development of triggered activity and myocyte contraction were

used as endpoints, the dose response curve of BPS was nonmonotonic, which was similar to

BPA (Liang, Gao et al. 2014). BPS and BPA showed similar efficacy and potency, as well as

the same most efficacious dose (10-9 to 10-8 M). Our findings contrast with the lower potency

of BPS than BPA described in other studies. For EDCs, nonmonotonic dose response curves

have been commonly observed, and show important implication for toxicity assessment

(Vandenberg, Colborn et al. 2012). In a previous study, our lab showed that the BPA’s

nonmonotonic dose response in cardiac myocytes is the result of multiple monotonic

responses on individual elements of Ca2+ handling. It is a combined effect of its inhibitory effect on Ca2+ influx through L-type Ca2+ channel and opposing stimulatory effect on SR

Ca2+ release and reuptake (Liang, Gao et al. 2014). This mechanism may provide clues to

explain BPS’ nonmonotonic dose response in the cardiac myocytes.

The responses of female and male rat hearts to low-dose BPS were strikingly different.

Female rat hearts have robust responses to acute BPS exposure at the whole heart, myocyte and protein levels; however, there were no detectable responses in male hearts when measuring the same endpoints. Notably, BPS did not enhance arrhythmic events including ectopic beats or ventricular tachycardia in male rat hearts under Iso-induced stress, and did not increase arrhythmic activities in isolated male rat ventricular myocytes. At the myocyte and protein levels from female hearts, acute BPS exposure altered Ca2+ handling and

phosphorylation of Ca2+ handling proteins in female rats, while these actions were not

observed in male rats. Our lab has previously demonstrated that such gender-specificity of

cardiac effects of estrogenic chemicals was caused by the opposing and counterbalancing

actions of two distinct ER signaling systems (Belcher, Chen et al. 2012). Thus, ER signaling

was stimulatory for Ca2+ handling and triggered activity development in both male and female rat ventricular myocytes, while ER signaling produced inhibitory effects. The balance between ER and ER signaling determined the response of the heart. Therefore, in

female rat hearts, estrogenic chemicals were pro-arrhythmic because of the dominance of

ER stimulatory signaling. In male rat hearts, though ER effect was present, its effects were

masked by ER inhibitory signaling, with the net effect resulting in no observable response.

Data in Figure 25 indicated that males’ lack of response to BPS may be explained by a

similar mechanism. ER activation with the selective agonist, DPN, demonstrated that in

male rat ventricular myocytes, the stimulatory effect of ER signaling was still intact. With

the blockade of ER using the selective blocker, MPP, ER signaling was revealed and BPS

presented a stimulatory effect on male rat ventricular myocytes.

In summary, this dissertation demonstrated that exposure to BPS at low-dose has rapid pro-arrhythmic effects on female but not male rat hearts; the underlying mechanisms likely involve ER signaling activation and myocyte Ca2+ handling alteration. The rapid cardiac

actions of BPS, measured using multiple endpoints, are very similar to BPA as described

earlier in Result 1. The similarity was also manifested at the cellular as well as molecular

mechanistic levels underlying the cardiac effects of these two chemicals. Taken as a whole,

our findings indicate that BPS, as well as other structurally-related BPA substitutes, may have

similar endocrine disrupting activities to BPA. The comprehensive evaluation of these

chemicals using biological and toxicological assays should be performed before they are used

in products in the consumer market.

Result 3. Probenecid alters mouse cardiac myocytes contractility by

affecting Ca2+ handling

3.1. Background and rationale

Probenecid was originally used to decrease renal tubular excretion of penicillin and

prolong its effects (Burnell and Kirby 1951; Meads, Knight et al. 1951). Later it, was also

developed to increase serum concentrations of several other antibiotics (Butler 2005). During

the early studies using probenecid, researchers found that probenecid had a strong uricosuric

effect; therefore it quickly became a primary treatment for gout. However, in recent years, the

clinical use of probenecid has decreased significantly with new therapies showing more

efficacy and better tolerance (Rider and Jordan 2010).

In the cardiovascular system, the effect of probenecid has not been fully investigated.

Collaboratively, The Rubinstein lab and our lab investigated the cardiac effects of probenecid

on the whole animal, isolated whole heart, and isolated ventricular myocyte levels with the

hypothesis that it can affect cardiac function. It was demonstrated by The Rubinstein lab that

probenecid increased myocardial contractility at both in vivo and isolated whole heart levels

in adult mice. Injection of 200 mg/kg probenecid into wildtype (WT) mice increased cardiac

contractility as measured via ejection fraction (EF) from echocardiography compared to

saline-injected mice. The inotropic effect was observed rapidly, within 5 minutes of i.v. injection and lasted for 1 hour. However, probenecid’s inotropic effect was not seen in

TRPV2-/- (knockout) mice at the same dose, and only showed half of peak value in

TRPV2+/- (heterozygous) mice. On the ex vivo whole heart level, it was shown that probenecid increased myocardial contractility as measured by the rate of left ventricle

100

pressure rise (+dP/dt) (Koch, Gao et al. 2012).

Probenecid has been identified to be a selective agonist of transient receptor potential

vanilloid 2 (TRPV 2) channels (Bang, Kim et al. 2007). It was demonstrated by Iwata’s

colleagues that cardiac-specific overexpression of TRPV2 could cause dilations of all

chambers in the mouse heart (Iwata, Katanosaka et al. 2003). However, the existence of

TRPV2 channels in myocardial tissues was not clearly elucidated. Dr. Rubinstein’s lab used

quantitative RT-PCR to test the mRNA of TRPV2 channels from hearts of WT, TRPV2+/-,

and TRPV2-/- mice. They identified a decrease in the expression levels of TRPV2 mRNA in

TRPV2+/- mouse hearts compared to WT. No expression was detected in TRPV2-/- mouse

hearts (Koch, Gao et al. 2012).

On the myocyte level, our lab examined the effect of probenecid on isolated mouse

ventricular myocytes. Fractional shortening was measured to evaluate the mechanical

function of myocytes upon exposure to probenecid. A blocker for TRPV2 channels,

ruthenium red, was used to evaluate the target for probenecid on the myocytes. [Ca2+]i was measured via confocal microscopic fluorescent imaging on the myocytes to study possible cellular mechanisms.

3.2. Probenecid increased isolated mouse ventricular myocyte contractility in a dose-dependent manner

In isolated mouse ventricular myocytes, our lab found that probenecid increased the contractility in a dose-dependent manner (Figure 26A). When exposed to 10-7 M probenecid

and stimulated at 0.5 Hz at room temperature (25 °C), myocyte fractional shortening (FS)

was increased from 6.9% to 9.0% (a fractional increase of 30.8 ± 1.4%, n = 6, P < 0.01). At 3

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Figure 26. Effect of probenecid on contractility of isolated mouse ventricular myocytes.

A. Example contraction traces of isolated mouse ventricular myocytes when exposed to 10−7

M probenecid. PROB, probenecid. B. Dose response curve of probenecid on myocyte fractional shortening increase. Data points are averages of 35 myocytes from 4 animals.

Sigmoidal non-linear regression was applied for fitting data points.

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Hz stimulation and 32 °C, probenecid increased FS from 10.1% to 13.9% (n = 4; P < 0.01).

The +dL/dt and -dL/dt were measured in isolated mouse ventricular myocytes. At room

temperature, an increase in maximum +dL/dt from 80.6 ± 12.3 to 112.5 ± 16.6 (n = 10, P <

0.01) and an increase in -dL/dt from 57.7 ± 7.4 to 77.6 ± 8.7 μm/s (P < 0.01) were observed.

To generate a dose response curve for the action of probenecid on myocytes, a series of

-10 -6 concentrations of probenecid from 10 to 10 M were examined. The EC50 for myocyte fractional shortening was 1.6 nM (Figure 26B). When pretreating myocytes with ruthenium red, which is a non-selective blocker of TRPV2 channels, probenecid's effect on myocyte contractility was completely blocked (Figure 27A and 27B).

Our lab also studied the effect of probenecid on Ca2+ transient in isolated myocytes. 10-7

M probenecid exposure caused a significant increase in Ca2+ transient amplitude (ΔF/F0)

from 3.35 ± 0.20 to 4.25 ± 0.25 (P < 0.001) (Figure 28A and 28B), consistent with the

myocyte fractional shortening increase recorded previously. The decay rate (Tau) of Ca2+ transient was not altered under 10-7 M probenecid exposure (Figure 28B).

3.3. Probenecid increased cytosolic Ca2+ in isolated mouse ventricular myocytes

The effects of probenecid on cytosolic Ca2+ and SR Ca2+ release was evaluated using

confocal microscopic imaging. In isolated mouse ventricular myocytes, 10-7 M probenecid

exposure for 5 minutes caused a marked increase in SR Ca2+ release which was measured by

Ca2+ spark frequency (Figure 29A and 29B). The spark frequency was increased from 1.45 ±

0.09 to 2.99 ± 0.24, which was about two-fold on average. The fluorescent intensity of line scanning was calculated to measure cytosolic Ca2+ levels in myocytes. As shown in Figure

30A and 30B, there was a gradual increase in cytosolic Ca2+ concentration with 10-7 M

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Figure 27. Blockade effect of ruthenium red on probenecid’s alteration of myocyte contractility. A. Example contraction traces of isolated mouse ventricular myocytes when exposed to 10−7 M probenecid with 10−6 M ruthenium red pretreatment. B. Average data on myocyte fractional shortening (FS) with 10−6 M ruthenium red pretreatment under control and

10−7 M probenecid exposure. n = 32 myocytes of 4 hearts. NS, not significant.

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Figure 28. Effect of probenecid on Ca2+ transient in isolated mouse ventricular myocytes.

A. Example Ca2+ transient traces under field stimulation upon 10−7 M probenecid treatment. B.

Average data on Ca2+ transient amplitude F/F0 (left) and decay time constant tau (right) under control and 10−7 M probenecid treatment. n = 16 myocytes of 4 hearts. *, P < 0.001, NS., not significant.

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Figure 29. Effect of probenecid on Ca2+ spark in isolated mouse ventricular myocytes. A.

Example images of confocal microscopic line scan of isolated mouse ventricular myocyte

under control and under exposure to 10−7 M probenecid for 5 minutes. A heat map is shown to indicate the intensity of F/F0 with values from 0 to 2. B. Average data on the frequency of

Ca2+ spark in the myocytes under control and under exposure to 10−7 M probenecid for 5

minutes. n = 15 myocytes of 5 hearts. *, P < 0.001.

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probenecid treatment. The cytosolic Ca2+ began to increase after 1 minute of probenecid

treatment and reached a peak at around 5 minutes (Figure 30A). At steady state, cytosolic

Ca2+ was increased 2.5 ± 0.2 fold compared to control (Figure 30B). With ruthenium red (10-6

M) pretreatment of the myocytes, the increase of cytosolic Ca2+ level by probenecid was

completely blocked (Figure 30C).

In order to find out the cause of cytosolic Ca2+ increase in isolated mouse ventricular myocytes with probenecid treatment, whole-cell patch clamp was used to measure the Ca2+ influx through the cell membrane. As shown in Figure 31A, 10-7 M probenecid did not trigger

any significant transmembrane Ca2+ influx in the myocytes. There was no measurable inward

Ca2+ current detected when myocytes were exposed to 10-7 M probenecid and held at -70 mV.

However, a robust L-type Ca2+ current was observed when depolarizing the myocyte

membrane from −70 mV to +10 mV (Figure 31B). This suggests that increased SR Ca2+ release plays a major role in increasing the cytosolic Ca2+ level in the isolated myocytes.

When SR Ca2+ content was emptied with thapsigargin, which is a SERCA blocker, the change

in cytosolic Ca2+ by probenecid was fully abolished (Figure 31C and 31D).

Discussion 3

At the cellular level, probenecid increased contractility of isolated mouse ventricular

myocytes. Probenecid increased the amplitude of Ca2+ transients in stimulated myocytes and

also increased cytosolic Ca2+ levels in quiescent myocytes. No measurable inward Ca2+

current was detected upon exposure to probenecid, suggesting that the probenecid-triggered

increase in cytosolic Ca2+ levels was not directly caused by Ca2+ influx into the myocyte.

Probenecid markedly increased Ca2+ spark frequency in isolated myocytes. When emptying

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Figure 30. Effect of probenecid on cytosolic Ca2+ level in isolated mouse ventricular

myocytes. A. Time courses of cytosolic Ca2+ levels in isolated ventricular myocytes with and

without 10−6 M ruthenium red (RR) pretreatment under exposure to 10−7 M probenecid. Data

are normalized to time 0. B and C. Average data on myocyte cytosolic Ca2+ levels under control and 10−7 M probenecid treatment without (B) and with (C) RR pretreatment. n = 36

myocytes of 4 hearts. *, P < 0.001, NS., not significant.

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Figure 31. Probenecid’s effect on cytosolic Ca2+ was caused by SR Ca2+ release. A. Patch

clamp recording of a myocyte showing no Ca2+ influx under 10−7 M probenecid exposure. B.

Patch clamp recording of the same myocyte of (A) showing L-type Ca2+ current elicited by

depolarization voltage step to +10 mV. The holding potential for myocytes was -70 mV. C.

Example confocal images of isolated mouse ventricular myocyte pretreated with 10−6 M thapsigargin (TG), before and after 10−7 M probenecid treatment. D. Average data on myocyte

cytosolic Ca2+ levels in myocytes pretreated with 10−6 M thapsigargin, under both control and

10−7 M probenecid treatment conditions. n = 15 myocytes of 4 hearts. #, not significant.

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the SR Ca2+ store with a SERCA blocker, the effect of probenecid on cytosolic Ca2+ levels

was completely abolished. These results indicated that enhanced SR Ca2+ release was more

likely to play the major role in probenecid-induced cytosolic Ca2+ increase.

Probenecid was recently identified as a potent and selective agonist of transient receptor

potential vanilloid 2 (TRPV2) channel (Bang, Kim et al. 2007). TRPV2 is a member of the

thermo TRP channel family. It is a Ca2+ selective channel sensitive to noxious heat (>52 °C).

TRPV2 activation has been shown to induce Ca2+ influx in dorsal root ganglia neuron cells

(Shibasaki, Murayama et al. 2010). The presence of TRPV2 channels in cardiac tissues is described by our collaborator, the Rubinstein lab (Koch, Gao et al. 2012). Based on earlier literature and our results, it is hypothesized that probenecid's stimulatory effects on cytosolic

Ca2+ levels and myocyte contractility in isolated mouse ventricular myocytes involved

TRPV2 activation, which could be supported by the fact that ruthenium red (RR), which is a

blocker of TRPV2 channels, could fully abolish probenecid's effects on both contractility and

cytosolic Ca2+ levels. RR has been well recognized as a general antagonist for TRPV channels through blocking aqueous pores in the channels (Vriens, Appendino et al. 2009).

However, further experiments with TRPV2 knockout mice need to be done for verification.

The mechanism underlying the effect of probenecid on SR Ca2+ release is not completely

understood. It is hypothesized that probenecid activated TRPV2 channels, resulting in a local

increase in Ca2+ levels and triggering the activation of nearby ryanodine receptors. It was

shown by the Rubinstein lab that probenecid did not affect phosphorylation of either phospholamban or ryanodine receptors, indicating a direct action of probenecid on cellular

Ca2+ content, which increased the Ca2+ release at a beat-to-beat basis, resulting in enhanced

110 contractility of the myocytes (Koch, Gao et al. 2012).

Probenecid, a FDA-approved drug, has been used for decades in a variety of clinical scenarios. The Rubinstein lab found that probenecid increased contractility in both in vivo mouse heart, as determined by echocardiography, and Langendorff ex vivo hearts (Koch, Gao et al. 2012). Our lab studied the effect of probenecid on the isolated myocytes and the potential cellular mechanism behind its action. We collaboratively described the positive inotropic properties of probenecid which were previously not recognized. Our results obtained from isolated myocytes were consistent with their data in the time frame (5 minutes in all cases) which argues convincingly against a significant systemic increase in adrenergic drive as the main cause for the positive inotropic action. TRPV2 knockout mice had absolutely no change in cardiac function after exposure to probenecid, suggesting the activation of TRPV2 receptors is the likely cause for increased myocardial contractility by probenecid (Koch, Gao et al. 2012). Our findings have direct implications for cardiac disease patients who are currently receiving probenecid for treating gout. With the concern raised by our study, the clinical use of probenecid in cardiac disease patients requires a thorough analysis in terms of its mechanisms of action, dosing regimen, targets, as well as potential off-target effects.

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Chapter IV: Conclusions and Significance

The goals of this dissertation are to investigate the impact of environmental endocrine disrupting chemicals on the heart with a focus on cardiac arrhythmogenesis and to understand the underlying cellular and molecular mechanisms. As it has been demonstrated, BPA, a widely used plasticizing EDC found in consumer products, is pro-arrhythmogenic in female rodent cardiomyocytes through a mechanism involving PKA and CAMKII signaling cascades to affect the Ca2+ handling processes. Also, a popular substitute agent for BPA in plastics and thermal papers, BPS, was found to rapidly impact female rodent hearts at the organ, cellular and protein levels. The BPS effects were strikingly similar to BPA’s pro-arrhythmic effects in the heart at the equal dose range. In addition to the central goal of the dissertation, probenecid, a FDA-approved drug for gout treatment and antibiotic adjuvant therapy, was evaluated for its cardiac actions. It was demonstrated that probenecid had positive inotropic properties which were previously not recognized on cardiomyocytes, and that its effect was mediated by

TRPV2 channels via alteration of intracellular Ca2+ handling.

Cardiovascular disease is a major health problem in the world; it is one of the top contributors to morbidity and mortality in humans (Lloyd-Jones, Adams et al. 2010). While the genetic, physiological, as well as pathophysiological causes for cardiovascular disease are intensively studied, less is known concerning the impact of environmental chemicals. In this dissertation, the cardiac impact of EDCs including BPA and BPS was evaluated in detail, significantly enhancing our understanding of the cellular and molecular mechanisms explaining EDCs’ actions in the cardiovascular system. Previous epidemiological studies

112 suggested an association between BPA exposure in adult populations and cardiovascular diseases including heart attack, angina, and coronary artery disease (Lang, Galloway et al.

2008; Melzer, Rice et al. 2010; Melzer, Gates et al. 2012). At doses relevant to human-exposure, experimental studies on laboratory animals indicate that BPA exposure, both acutely and chronically, can affect the normal function of cardiovascular system function and increase the susceptibility for cardiovascular diseases including hypertension, cardiac arrhythmias, remodeling of heart and atherosclerosis. The cellular and molecular mechanisms behind BPA’s cardiovascular impact are being investigated by researchers, though the available data are not convincing. Based on the studies in this dissertation, in female rat cardiomyocytes, PKA signaling and CAMKII signaling are the two major pathways mediating low-dose BPA’s rapid impact on myocyte arrhythmogenesis. Rapid activation of

PKA increases phosphorylation of RyR and enhances its probabilities of opening, resulting in elevated SR Ca2+ leak. CAMKII activation results in increased PLN phosphorylation, which causes the release of PLN inhibition on SERCA and enhances SR Ca2+ reuptake. It was shown that these two pathways were indispensible for BPA-promoted triggered activities in cardiomyocytes. Previous studies have indicated that BPA’s cardiac effects were likely mediated by alteration of cardiac protein expression levels, oxidative stress, ion channel inhibition or activation, as well as genome modifications. Our studies provide a novel and distinct cell signaling mechanisms to describe BPA’s acute cardiac effects, which enhances our understanding of the mechanisms underlying EDCs’ actions. Though there is still some controversy, BPA environmental exposure can potentially be a contributor to increasing risks for cardiovascular diseases, necessitating further assessment of the potential cardiac toxicity

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of BPA and related estrogenic EDCs.

BPS is a substitute chemical replacing BPA in many consumer goods in the market,

leading to high exposures for humans (Liao and Kannan 2013). However, it is not clear

whether BPS has a better safety profile than BPA. Current knowledge on BPS’ biological

activities and potential toxic effects on humans is very limited. Only a few epidemiological

and experimental studies are available, and mechanistic studies underlying BPS’ actions are

scarce. This dissertation reported a female-specific action of low-dose BPS on the whole

heart and cardiomyocyte levels, which is strikingly similar with BPA’s pro-arrhythmic effects

in the heart (Yan, Chen et al. 2011). These results are of high significance because they

highlight the notion that BPS and other BPA substitutes are not necessarily safer than BPA for

human health.

As a separate study, this dissertation significantly advances our knowledge of a

traditional well-tolerated drug probenecid and its cardiac impact. Having been used for

decades, probenecid is seen in various clinical scenarios such as antibiotic adjuvant therapy

and gout (Robbins, Koch et al. 2012), but its effects on the heart were not thoroughly

investigated. Probenecid has limited water solubility, so when orally administered it is readily

absorbed into blood and binds to plasma proteins, particularly albumin (Cunningham, Israili

et al. 1981). In its usage for gout treatment, probenecid is administered 250 mg twice a day to a maximum 3 g per day (Hilaire and Wozniak 2010). When applied as an antibiotics adjunct agent, its therapeutic dose is 1 – 2 g per day. Serum probenecid concentration is shown to be from 35.3 to 69.6 mg/mL when used at standard therapeutic dose range (Robbins, Koch et al.

2012), equivalent to 120 to 240 mM probenecid. According to the dose response curve in our

114 cardiomyocyte study, at these concentrations the mechanical function of mouse ventricular myocyte such as fractional shortening is significantly increased (30%). Data from our collaborator’s lab indicated that probenecid enhanced mouse myocardium contractility both in vivo and on the ex vivo whole heart, but did not affect cardiac electrical conduction (Koch,

Gao et al. 2012). No supraventricular or ventricular arrhythmias were observed at relevant doses, and no change in conduction intervals was recorded. These results are interesting and potentially important, since commonly used inotropic drugs increase the likelihood of arrhythmias. Digoxin may cause atrial tachycardia and AV block; dopamine, dobutamine and isoproterenol can produce ventricular and supraventricular arrhythmias (Williamson,

Thrasher et al. 1998; Bouvy, Heerdink et al. 2000). Thus, probenecid may be of clinical use as a positive inotropic drug for patients lacking the arrhythmogenic side effects, though a thorough analysis of its target and off-target effects is necessary. Also, this study has immediate implications for cardiac disease patients who also take probenecid for gout therapy.

Future directions

In the dissertation, it was demonstrated that BPA’s pro-arrhythmogenic effects in female cardiomyocytes were mediated by PKA and CAMKII signaling via ERβ. The mechanisms involved alteration of Ca2+ handling and enhanced SR Ca2+ leak. However, there are still questions to be answered in this mechanistic study. As shown in the study, RyR and PLN were selectively phosphorylated by PKA and CAMKII. Such target-specific phosphorylations by kinases are likely caused by formation of kinase/anchoring protein/target protein complexes, by which kinases are able to regulate their target activities both temporally and

115

spatially (McConnachie, Langeberg et al. 2006; Maier and Bers 2007).

Co-immunoprecipitation, will be able to pull down the working complex by targeting RyR or

PLN and dissect the components of the complex to elucidate how the target-specificity was

achieved in cardiomyocytes under BPA exposure. This will provide insights to understand the

differences between BPA signaling and adrenergic signaling, and how they interact to induce

arrhythmias in the heart.

Next, we used rat hearts in this study to investigate the cardiac actions of BPA, due to the

common physiology and pathophysiology of hearts between rodents with humans. However,

since large animals such as pigs and dogs are even more similar to human cardiology, for

instance, in terms of cardiac electrophysiology, we will study the large animals’ responses to

BPA exposure in future. Indeed, pioneering studies on dogs from our laboratory discovered

the rapid change of action potential duration as well as action potential morphology when

isolated cardiomyocytes were treated with BPA. Moreover, as we observed in this dissertation

study, BPA at environmentally relevant doses did not trigger detectable arrhythmia in normal

rodent hearts, but it exacerbated ventricular arrhythmias in female rat hearts when stress or

ischemia is present (Yan, Chen et al. 2011; Yan, Song et al. 2013). These results indicate that

BPA alone at common environmental exposure levels may not be sufficient to induce

clinically significant cardiovascular diseases in healthy individuals; while in populations

predisposed to existing pathophysiological conditions, BPA exposure could be more

dangerous. Therefore, we will use cardiac disease animal models to simulate pathological

conditions in humans, and evaluate BPA’s effects under various disease settings.

BPS, a new BPA substitute chemical used in plastics and thermal paper, was studied in

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this dissertation. It was found that BPS showed parallel pro-arrhythmic effects with BPA at

the same doses in rat cardiomyocytes. It is worth noting that there are many more BPA

derivatives and substitutes in the consumer market whose safety profiles are not established,

posing a health threat. For instance, tetrabromobisphenol A (TBBPA) is a brominated BPA

derivative and the most commonly used flame retardant. The estimated global market

demand for TBBPA exceeded 120,000 metric tons in 1999 (de Wit 2002). As a result, TBBPA

became a prevalent contaminant found in the environment, including river, sewage, air, and

wildlife (Watanabe, Kashimoto et al. 1983; Sjodin, Carlsson et al. 2001; Alaee, Arias et al.

2003; Morris, Allchin et al. 2004; Zhang, Luo et al. 2009). The wide use of TBBPA increases

concerns over its potential toxic effects on humans and wildlife (Johnson-Restrepo, Adams et

al. 2008). Indeed, it was found that TBBPA mimics estrogens and interferes with normal

development and reproduction of animals. In zebrafish, TBBPA affected egg production,

survival rate and caused developmental lesions (Kuiper, van den Brandhof et al. 2007; Song,

Liang et al. 2014). TBBPA was also shown to disrupt thyroid hormone signaling, alter

estrogen synthesis and damage sperm DNA integrity (Honkisz and Wojtowicz 2014; Zatecka,

Castillo et al. 2014; Zhang, Xu et al. 2014). However, with limited literature available, the

biological effects of TBBPA on the heart are currently unknown. The cardiac effects of

TBBPA will need to be evaluated using whole heart and isolated cardiomyocytes from

rodents and potentially investigate the underlying cellular and molecular mechanisms, which will provide important information on the toxicological profile of TBBPA as well as other halogenated BPA derivatives.

The potential adverse impact of EDCs on the heart was a focus of this dissertation.

117

Recent studies have shown that the toxicities of EDCs such as BPA could possibly be prevented by medical plant extracts tested in renal and hepatic cell lines, which was mediated partly by cytochromes P450 modulation (Gasnier, Laurant et al. 2011). It was reported that ginsenosides protected against BPA-induced cell cytotoxicity of sertoli cells mediated by inhibiting ERK1/2 phosphorylation and enhancing cellular antioxidation (Wang, Hao et al.

2012). Also, a dietary supplement, chitosan oligosaccharide lactate (ChOL) was found to protect the human gingival fibroblasts (HGFs) against DNA double-strand breaks caused by

BPA-based dental adhesive (Szczepanska, Pawlowska et al. 2011). These studies point to the potential preventative and protective effects of several natural chemicals and dietary supplements against the adverse impact of BPA on human health. Our future studies will explore the possible protective methods using these natural or dietary compounds to alleviate the pro-arrhythmogenic effects of BPA in the heart.

This dissertation also described the inotropic properties of probenecid, a FDA-approved drug for gout, on the cardiomyocytes. Our collaborator also reported that both in vivo and on the ex vivo hearts, probenecid increased myocardial contractility. The fact that it did not affect electrical conduction or promote ventricular arrhythmias on the whole heart makes it a candidate for inotropic medication for heart failure patients. Currently, acute heart failure is mostly treated with vasodilators to reduce afterload, and inotropic drugs to increase myocardial contractility (Gheorghiade, Zannad et al. 2005). For the second method, sympathomimetics such as dobutamine are commonly used medications, though they all have potential side effects including arrhythmogenesis and inducing apoptosis to the myocytes

(Williamson, Thrasher et al. 1998; Burger, Elkayam et al. 2001; Singh, Xiao et al. 2001;

118

Goldhaber and Hamilton 2010). As a result, the outcomes for inotropic therapy are not as

good as vasodilation, sometimes leading to poor prognosis (Felker and O'Connor 2001;

Abraham, Adams et al. 2005). As demonstrated, probenecid increased isolated myocyte

fractional shortening and enhanced myocardial contractility on Langendorff perfused whole

heart and in vivo hearts of mice, with no cardiac arrhythmic events noted. To evaluate the

potential therapeutic usage of probenecid on acute heart failure, we will test its effect using a

cardiac diseased mouse model. We also need to measure its cell injurious (cytotoxic and

apoptotic) properties on cardiomyocytes under the pathological condition. Another direction

is to elucidate the molecular mechanisms underlying probenecid’s cardiac actions. It is

known that probenecid is a selective agonist for TRPV2 channels, and TRPV2 is present in

the cardiac tissues especially in the ventricles, but we have not been able to directly measure

the current of TRPV2 on cardiomyocytes upon probenecid application. According to our data,

probenecid did not trigger detectable transmembrane Ca2+ influx but increased [Ca2+]i level;

likely because that TRPV2 is located intracellularly. We will use immunofluorescent imaging

to identify the location of TRPV2 in cardiomyocytes and examine how it participates in myocyte Ca2+ handling process using TRPV2-ablation animal models.

119

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Appendix: Publications and Abstracts

1. Publications

Koch, S. E., Gao, X., Haar, L., Jiang, M., Lasko, V. M., Robbins, N., Cai, W. F., Brokamp,

C., Varma, P., Tranter, M., Liu, Y., Ren, X. P., Lorenz, J. N., Wang, H-S., Jones, W. K.

and Rubinstein, J. (2012). "Probenecid: Novel use as a non-injurious positive inotrope

acting via cardiac TRPV2 stimulation." Journal of Molecular and Cellular Cardiology

53(1): 134-144.

Manning, J. R., Perkins, S.O., Sinclair, E. A., Gao, X., Zhang, Y., Newman, G., Pyle, W. G.

and Schultz, J. E. (2013). "Low molecular weight fibroblast growth factor 2 signals via

protein kinase C and myofibrillar proteins to protect against post-ischemic cardiac

dysfunction." Am J Physiol Heart Circ Physiol 304(10): H1382-96.

Singh, V. P., Rubinstein, J., Arvanitis, D. A., Ren, X., Gao, X., Haghighi, K., Gilbert, M., Iyer,

V. R., Kim, H., Cho, C., Jones, K., Lorenz, J. N., Armstrong, C. F., Wang, H-S., Gyorke,

S. and Kranias, E. G. (2013). "Abnormal calcium cycling and cardiac arrhythmias

associated with the human S96A genetic variant of histidine-rich calcium binding

protein." J Am Heart Assoc 2(5): e000460.

Gao, X., Liang, Q., Chen, Y. and Wang, H-S. (2013). "Molecular mechanisms underlying the

rapid arrhythmogenic action of bisphenol A in female rat hearts." Endocrinology

154(12): 4607-17.

Liang, Q., Gao, X., Chen, Y., Hong, K. and Wang, H-S. (2014). "Cellular mechanism of the

non-monotonic dose response of bisphenol A in rat cardiac myocytes." Environ Health

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Perspect 122(6): 601-8.

Gao, X. and Wang, H-S. (2014). "Impact of bisphenol A on the cardiovascular system –

epidemiological and experimental evidences and molecular mechanisms." Int J Environ

Res Public Health 11(8):8399-413.

Gao, X., Ma, J., Chen, Y. and Wang H-S. (2015). "Rapid responses and mechanisms of action

for low-dose bisphenol S on ex vivo rat hearts and isolated myocytes: evidence of

female-specific pro-arrhythmic effects." Accepted by Environ Health Perspect

1. Abstracts

Liang, Q., Gao, X., Chen, Y. and Wang, H-S. (2012). "Elucidation of the Cellular Mechanism

Underlying the Inverted-U Shaped Dose Response of BPA in Female Hearts." NIEHS

BPA Grantee Meeting, NIEHS (National Institute of Environmental Health Sciences)

campus, Research Triangle Park, NC.

Gao, X., Koch, S. E., Jiang, M., Robbins, N., Cai, W. F., Varma, P., Tranter, M., Jones, W.

K., Wang, H-S. and Rubinstein, J. (2012). "TRPV2 Regulates Cardiomyocyte Function

via Altering Ca2+ Cycling." AHA Basic Cardiovascular Sciences (BCVS) 2012 Scientific

Sessions, Hilton New Orleans Riverside, New Orleans, LA.

Gao, X., Liang, Q., Chen, Y. and Wang, H-S. (2013). "Elucidation of the Signaling

Mechanism Underlying BPA’s Rapid Arrhythmogenic Action in Female Hearts." The

Endocrine Society’s 95th Annual Meeting and Expo, Moscone Center, San Francisco,

CA.

Liang, Q., Gao, X., Chen, Y. and Wang, H-S. (2013). "Cellular Mechanism of the

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Non-monotonic Dose Response of BPA in the Heart". The Endocrine Society’s 95th

Annual Meeting and Expo, Moscone Center, San Francisco, CA.

Gao, X., Ma, J., Chen, Y. and Wang, H-S. (2014). "Evaluation of the Rapid Effects of

Bisphenol S (BPS) on the Heart: Impact on Arrhythmogenesis and Cardiac Calcium

Handling." (Oral Presentation) The Endocrine Society’s 96th Annual Meeting and Expo,

McCormick Place West, Chicago, IL.

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