TRP Channels in Cardiovascular Disease

University of Connecticut University of Connecticut

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Doctoral Dissertations University of Connecticut Graduate School

5-8-2020

TRP Channels in Cardiovascular Disease TRP Channels in Cardiovascular Disease

Albert Yu University of Connecticut - Storrs, [email protected]

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TRP Channels in Cardiovascular Disease

Albert S. Yu, PhD

University of Connecticut, 2020

Abstract

Transient receptor potential (TRP) channels are evolutionarily conserved ion

channels that have been implicated in a wide range of physiological and

pathophysiological responses. As versatile ion channels that are permeable to calcium,

the exact nature of these ion channels have been furiously studied since its initial

discovery in Drosophila. Many TRP channels are thought to be gated by PIP2, a well-

known membrane signaling molecule. Here we show that membrane potential alters PIP2

in such a way to reduce TRPM7 activity, presumably through PIP2 depletion. This novel

mechanism of TRPM7 regulation gives us a clearer picture into a complicated protein that

has been implicated in processes ranging from embryonic development to cancer.

Furthermore, we show how TRP channels are involved in the development and

progression of various cardiovascular diseases. Our research implicates the oxidative

stress activated channel TRPM2 in the progression of atherosclerosis, with data pointing

to its role in driving inflammation by increasing circulating myeloid cell populations. We

also show that the bifunctional channel-enzyme TRPM7 plays a deleterious role in the

cardiac fibrogenesis cascade in hypertensive heart failure, and deletion of Trpm7

specifically in the cardiac fibroblast is protective against negative cardiac remodeling. My

research demonstrates not only how TRP channels are important mediators of

cardiovascular disease, but also how they are regulated at a basic molecular level.

i


Albert S. Yu

B.A. University of California Berkeley, 2011

A Dissertation

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Doctor of Philosophy

at the

University of Connecticut

2020

ii

Copyright by

Albert S. Yu

2020

iii

APPROVAL PAGE

Doctor of Philosophy Dissertation


Presented by Albert S. Yu, B.A.

Major Advisor ___________________________________________________________________ Lixia Yue, PhD Associate Advisor ___________________________________________________________________ Kevin Claffey, PhD Associate Advisor ___________________________________________________________________ Kimberly Dodge-Kafka, PhD Associate Advisor ___________________________________________________________________ Linda Shapiro, PhD

University of Connecticut 2020

iv

Acknowledgments I would like to thank my friends and family both in Connecticut and California for

supporting me through this journey. I also would like to thank my committee members, my advisor Dr. Lixia Yue, past and current lab members in particular Dr. Yanlin He, Dr. Jianlin Feng, and Dr. Zhichao Yue, Dr. Hector Leonardo Aguila, the MD/PhD program especially Dr. Carol Pilbeam and Tracy Dieli, everyone in the Cell Biology and Cardiology Departments, and the Graduate School for helping me develop into the researcher and person that I am today. Finally, I would like to thank the American Heart Association for providing a gracious Predoctoral Fellowship.

v

Table of Contents Feature Page Approval Page .................................................................................................... iii. Acknowledgements ............................................................................................. iv. Table of Contents ................................................................................................. v. List of Figures ..................................................................................................... vi. Chapter and Title Page Chapter 1: Introduction ................................................................................... 1

Cardiovascular disease as a public health crisis ................... 2

A TR(i)P in ion channels ....................................................... 7 Chapter 2: Membrane potential regulation of TRPM7 .................................. 13 Chapter 3: TRPM2 modulation of inflammation and CVD ............................ 34 Chapter 4: Targeting TRPM7 to reverse cardiac fibrosis .............................. 52 Chapter 5: pH regulation of the gatekeeper, Orai ......................................... 60 Chapter 6: Conclusions, Discussion, and the future TR(i)Ps ........................ 72 Chapter 7: References ................................................................................. 75

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List of Figures Page Figure 1.1: A phylogenetic tree of TRP channels .......................................... 11

Figure 1.2: Membrane topology of TRPs ...................................................... 12

Figure 2.1A: Hyperpolarized membrane potential inactivates the

TRPM7 channel .......................................................................... 24

Figure 2.1B: TRPM7 inactivation shows time dependent inactivation

and recovery ............................................................................... 25

Figure 2.1C: Recovery after inactivation approaches near maximum ............. 26

Figure 2.1D: Hyperpolarized membrane potential by step protocol also

inactivates the TRPM7 channel .................................................. 27

Figure 2.1E: Preservation of intracellular contents by perforated patch technique

recapitulates whole cell results ................................................... 28

Figure 2.2: Dynamic PIP2 changes with membrane potential ........................ 29

Figure 2.3: Addition of exogenous PIP2 does not prevent TRPM7

inactivation .................................................................................. 31

Figure 2.4: Divalent ions are not likely responsible for low voltage induced

change ........................................................................................ 32

Figure 2.5: Model of TRPM7 inactivation by low voltage .............................. 33

Figure 3.1: Trpm2 deletion reduces atherosclerosis ..................................... 42

Figure 3.2: Trpm2 deletion protects against myocardial infarction ................ 43

Figure 3.3A: Flow cytometry process .............................................................. 45

Figure 3.3B: Trpm2 deletion does not alter myeloid subpopulations

in the healthy state ...................................................................... 46

vii

Figure 3.3C: Systemic changes in circulating myeloid cells

during atherosclerosis ................................................................. 47

Figure 3.3D: Myocardial infarction does not alter peripheral

myeloid populations .................................................................... 48

Figure 3.4: TRPM2 is expressed in bone marrow progenitor cells ................ 49

Figure 3.5: Model of TRPM2 induced inflammation in myeloid cells ............. 50

Figure 3.6: Myocardial specific, inducible deletion of Trpm2 to study

atherosclerosis and myocardial infarction ................................... 51

Figure 4.1: Deletion of Trpm7 attenuates fibrosis and improves

heart function .............................................................................. 57

Figure 4.2: Fibroblast specific deletion of Trpm7 recapitulates

protective effects ......................................................................... 58

Figure 4.3: Schematic of TRPM7, the chanzyme .......................................... 59

Figure 5.1: Acidic and basic external pH on Orai1/STIM1 channels ............. 66

Figure 5.2: Effects of pH on Orai2 and Orai3 ................................................ 67

Figure 5.3: pH effects on Orai1 mutants ....................................................... 68

Figure 5.4: Effects of internal pH on Orai1/STIM1 ........................................ 69

Figure 5.5: H155 as the intracellular pH sensor of Orai1/STIM1 ................... 70

Figure 5.6: Effects of external calcium on pH sensitivity of Orai1/STIM1 ...... 71

1

CHAPTER 1: INTRODUCTION TO TRP CHANNELS IN CARDIOVASCULAR DISEASE

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Cardiovascular disease as a public health crisis

By 2030, 44% of the US population will be afflicted by cardiovascular disease

(CVD)1. Currently, CVD is the leading cause of death not only in the US but also

worldwide. The phenomenon in developed countries is driven by lifestyle factors,

particularly diet and a sedentary lifestyle. In developing countries, this trend is also taking

place due to reduced morbidity and mortality from infectious disease and a shift to a

Western lifestyle. Thus, a main priority today in healthcare and research today is finding

ways to reduce CVD morbidity and mortality.

Cardiovascular disease encompasses a wide array of diseases, including

peripheral artery disease to congenital as well as acquired heart disease 2. The most

common subtypes of CVD stem from a process known as atherosclerosis. From Greek

and Latin origins, “atherosclerosis” was first coined due to its appearance as a “porridge-

like hardening.” Biologically, an atherosclerotic plaque is composed of many different cell

types reacting to lipid deposition in the arterial walls 3. Surprisingly, studies have shown

that signs of atherosclerosis begin in adolescence, and progresses with age 4.

The initial events of atherosclerosis are thought to begin by abnormal blood flow

and stress to the arterial wall 5. The disturbed flow can be caused by increased viscosity

of the blood, for instance, by an increase in lipid content. In regions of abnormal blood

flow, endothelial cells express cell surface adhesion molecules while concurrently

allowing the entry of lipoprotein particles from the bloodstream into the arterial wall 6. The

lipoprotein particles in the arterial wall are engulfed and eliminated by resident arterial

cells, such as fibroblasts, macrophages, and endothelial precursor cells. However, during

continued deposition of lipoprotein particles into the arterial wall, the engulfing cells

3

become overloaded and insufficiently eliminate lipid particles, leading to apoptosis and

release of inflammatory cytokines. These events lead to the proliferation of resident cells,

as well as the recruitment of circulatory cells into the arterial wall. However, as lipid

deposition continues, these plaques fail to completely resolve, leading to a vicious cycle

of inflammation and plaque growth 7.

A fundamental question exists as to what dictates macrophages in atheroma

lesions to secrete inflammatory cytokines. There is a widely held belief that once

macrophages enter lesion areas and engulf oxidized lipids, the macrophages convert to

foam cells that are able to generate both a protective and inflammatory reaction 8. The

balance between inflammation and resolution must be finely maintained 9 10.

Atherosclerosis is thought to be an inflammatory process by nature. Inflammation

of the arterial wall, with concomitant recruitment of immune cells and production of

inflammatory cytokines, has long been considered as critical in the disease process.

Patients with cardiovascular disease have elevated markers of inflammation such as

circulating IL-1β and CRP. Thus, general targets of inflammation, such as IL-1β blockers,

used to treat autoimmune inflammatory disorders, have recently been considered for use

for possible treatment of atherosclerosis 11 12 13. Interestingly, data showing the benefits

of IL-1beta blockers in autoimmune disorders also shows unexpected improvements in

cardiovascular outcomes 14.

Another recent finding in atherosclerosis research has shed light into the systemic

response generated by atherosclerosis 15. In addition to the local plaque secreted factors,

there may be inflammatory cytokines that travel systemically to enhance the recruitment

of cells in an attempt to re-establish homeostasis. Furthermore, clinical data show

4

differences in the circulating monocyte populations in patients with cardiovascular

disease 16.This has been confirmed in animal models of atherosclerosis which show

increased circulating leukocytes, predominantly due to the increase in myeloid cells 17.

Furthermore, animal models have shown that these circulating myeloid cells adopt an

inflammatory state, as denoted by high expression of the inflammatory marker Ly6C,

which are also found in early atherosclerotic lesions 18. The presence of these myeloid

cells in early atheroma lesions suggests that these cells play a critical role in plaque

development and disease progression. Thus, there has been a strong interest in studying

cardiovascular disease from an immunological perspective.

As plaques grow, there are several ways blood flow can be reduced. First, the

plaque cap may become disrupted, leading to a piece of the plaque, known as an

embolus, to lodge in a smaller, downstream artery. In addition, the disrupted plaque may

cause an inflammatory reaction, or thrombus, to form at the site. Both cause a reduction

in diameter of the artery or small vessel. Furthermore, abundant plaques throughout the

circulatory system can cause reduced blood flow systemically. This leads to an increased

systolic pressure, which eventually wears down the heart and reduces blood flow to vital

organs. Thus, when blood flow is reduced, there are several main diseases that arise.

When atherosclerosis reduces blood flow to the extremities, symptoms of tingling and

pain may arise, leading to a condition known as peripheral artery disease. When blood

flow to critical arteries supplying the brain are reduced, stroke may result. Finally, when

the coronary arteries are blocked, myocardial ischemia or infarction (heart attack) may

arise.

5

Myocardial infarction is one of the major consequences of plaque buildup. Typically

affecting the main left anterior descending coronary artery that supplies blood to a major

of the left ventricle, coronary artery disease typically presents as a sharp, radiating pain

in the chest and shoulder region accompanied by shortness of breath. Depending on the

severity and extent of coronary blockage, the symptoms may resolve or may quickly

precipitate into ventricular arrhythmia and sudden cardiac death. Treatment strategies for

coronary artery disease range from treatment with drug regimens, such as aspirin and

anticoagulants to heart rhythm control agents such as beta blockers, to more invasive

procedures such as percutaneous coronary intervention, which involves catheter guided

stent placement or physical plaque disruption, or open heart surgery to revascularize

cardiac tissue from an existing blood supply using a graft, typically the left internal

mammary artery. After an initial heart attack, an acute inflammatory phase is required for

proper healing, followed by a resolution phase. However, prolongation of either of these

two phases can lead to detrimental outcomes and cardiac remodeling, which is

responsible for recurrence. Following acute intervention, long term monitoring and drug

treatment is precisely maintained in order to prevent recurrence. Unfortunately, for many

patients, an initial heart attack episode is likely not the last. In concordance with what is

seen clinically, a recent study found that myocardial infarction in mice accelerates the

inflammatory process of atherosclerosis, thus increasing susceptibility to another acute

cardiovascular episode 19.

Stroke is the second most common cause of cardiovascular disease related death.

In the case of stroke, an artery supplying a critical area of the brain is narrowed, leading

to reduced blood flow. As the brain is an organ that relies on constant blood supply, the

6

consequences of reduced or loss of blood flow and lead to irreversible, and often,

physically manifesting, outcomes. In cases of acute stroke, thrombolytics and

anticoagulants are the mainstays in terms of treatment options.

Treatment strategies for CVD can range from targeting subclinical atherosclerosis

to treating acute emergencies to managing long term recovery and repair. Primary

prevention involves modification of lifestyle factors, including diet and exercise. Early

stages of atherosclerosis can be detected by elevated blood pressure, and treatment

often involves relaxation of vessels, controlling heart rate, and reducing blood volume.

Subclinical atherosclerosis can be detected by coronary angiography and tests that

measure heart function or arterial disease in stress conditions. When atherosclerosis

presents as an acute cardiovascular event, such as peripheral ischemia, stroke, or heart

attack, acute treatment can include the use of thrombolytics, anticoagulation, or

percutaneous intervention, followed by treatment with blood thinners. Long term

treatment include anticoagulation, and for myocardial infarction, heart rate control. Both

myocardial infarction and stroke are driven by the same underlying cause-

atherosclerosis. As discussed above, many current therapies rely on symptomatic

treatment and prevention of another acute episode. However, there is a substantial need

for improvement within this field as cardiovascular disease has risen to be the top cause

of morbidity and mortality worldwide. In all these conditions, a common theme is the

presence of inflammation. Efforts to reduce or alter inflammatory signaling pathways in

human disease for the purposes of treatments have been successful to some extent.

However, there is a need to target more specific cells and pathways that cause

inflammation in cardiovascular disease. The morbidity and mortality as well as the high

7

rates of recurrence and poor prognosis are cause for concern. As many of the drugs

mentioned above mainly manage CVD, there is an urgent need to develop drugs to target

CVD before and/or during the symptom presentation, in order to halt or reverse disease.

A TR(i)P in ion channels

Ion channels are fundamental mediators of signaling. First hypothesized by

Hodgkin and Huxley and later confirmed by Katz and Miledi, ion channels serve as an

electrical conduit to mediate signaling 20. Transporters build charge, membranes store

charge, and ion channels allow a conduit for passive diffusion of ions and thus electricity.

Ions not only mediate signaling events on their own, but its movement also generates

changes in membrane potential that lead to downstream signaling events, for example in

voltage gated ion channels. One well known ion in modulating signaling events is calcium.

Calcium is a necessary cofactor/allosteric modulator in many enzymatic reactions

including the well-known regulatory regions in protein kinase C. In terms of gating, ion

channels can be activated in many additional ways. One well known example is the

activation of skeletal muscle contraction. Calcium entry through L-type calcium channels

in the cell membrane can activate the ryanodine receptors (RyR) on the sarcoplasmic

reticulum. This triggers the release of calcium from the sarcoplasmic reticulum store,

providing enough calcium for muscle contraction. Furthermore, low levels of calcium in

the endoplasmic reticulum can trigger the activation of calcium entry, known as calcium

release activation channels (CRAC) 21. Thus, calcium levels are finely maintained in a

resting cell and upon activation are meticulously increased and decreased in a space and

8

time dependent manner to allow for precise signaling. Any perturbations in this system

can ultimately lead to altered signaling or cell death.

One family of ion channels found from across species is the Transient Receptor

Potential (TRP) class of ion channels. TRP channels were originally identified in the

Drosophila melanogaster when Cosens and Manning discovered a mutant fly that

exhibited a transient response to light as compared to a sustained response in wild type

22. This led to the discovery of the first role of the TRP channel as a protein that mediates

the signal of light transduction. A couple decades later, Montell rescued the trp mutant

with its cDNA, and cloned the channel 23. The TRP channel was originally postulated to

have eight transmembrane domains based on hydrophobicity, with hydrophilic N and C

termini 24. In addition, the C termini was found to have 8-9 repeats. It is now known that

the mammalian TRP superfamily includes 28 subtypes, with related genes found in flies,

yeast, worm, and fish, suggesting conserved evolutionary roles (Figure 1.1.). The 28

mammalian Trp genes have been identified, in six subfamilies, consisting of TRPC

(canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (Ankyrin), TRPP (polycystic),

and TRPML (mucolipin), grouped mainly by their homology (Figure 1.2). Of note is that

these TRP channels lack a voltage sensor in S4. Furthermore, many channels are

permeable to calcium, a well-known signaling messenger. TRP channels are thought to

arrange in homo or heterotetramers consisting of 6 transmembrane domain subunits, and

playing roles ranging from thermosensation to hearing and sight. As such, TRP channels

are thought to be sensory channels, relying on different physical and chemical stimuli to

activate or inhibit the channel 25. Distinguishing components of each channel subtype are

the ion permeating S5-S6 transmembrane segments, as well as the N and C termini

9

domains. TRPM7, for instance, contains a C terminus domain that encodes a functional

kinase. As the understanding of the role of TRP channels progresses, a critical need

exists to identify modulators of TRP channel activity. However, the function of this kinase,

as well as its relation to ion channel activity, is being further investigated. Furthermore,

recent literature and analyses of various TRP channel structures may give additional

insight into the role of TRP channel in physiological or pathophysiological responses.

As different TRP channels were discovered, the activation mechanisms were also

brought into focus. Although the physiological activators of many TRP channels have not

been clearly characterized, the diverse responsiveness of TRP channels to sensory

stimuli indicate that TRP channels may be important in receiving different sensory

modalities and suggests their importance in regulating both physiological and

pathophysiological responses across species. For example, TRPV1-4 and TRPM3 are

activated by high temperature, and TRPM8, TRPA1, and TRPC5 are activated by low

temperature. TRP channels can also sense mechanical (for example shear and osmotic

stretch), chemical, nociceptive and lipid signals. TRPM2 can sense oxidative stress

signals like ADPR and NAD+. Furthermore, TRP channels are known to interact with other

TRPs as well as calmodulin, kinases and phospholipases 26. Some TRP channels have

ubiquitous expression throughout the human body while some channels are constitutively

active in overexpression systems. TRP channels are expressed in the heart, brain,

immune cells, kidney, bladder, digestive tract, among other organs. Thus, the study of

TRP channels will yield insight not only into the signals that activate them, but also into

how they may play a role in both human physiology and pathophysiology.

10

Recent research has elucidated the structure of many TRP channels. The super

resolution structures of these channels allows us to not only compare these channels, but

also give us a clearer picture into what activates or inhibits these channels, and how they

may potentially play a role in human disease. Though many chemical activators and

inhibitors have been identified, these chemicals lack specificity for any particular ion

channel. Structural analysis may allow us to design specific drugs to modulate channel

activity. Furthermore, one widely believed mode of gating is through phosphoinositide

signaling. Thus, structural studies will allow us to find where endogenous molecules such

as PIP2 binds to modulate channel activity 27. In addition to PIP2 binding, which signaling

pathways change PIP2 levels and the extent to which PIP2 changes or its metabolites can

be detected by TRP channels to modulate activity will be an important question to going

further. The goal of my research is to identify how TRP channels, in particular TRPM2

and TRPM7, are involved in cardiovascular disease by using animal models, and to

understand the basic mechanisms of TRP channel gating by PIP2.

11

Figure 1.1. A phylogenetic tree of human, Zebrafish, and Drosophila TRP channels. Sequence homology shows TRP channels in 6 subfamilies that comprise proteins with distinct channel properties. TRP channels are conserved from Drosophila to humans, indicating its importance in evolution and function. 28 mammalian Trp genes have been identified, in six subfamilies, consisting of TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (Ankyrin), TRPP (polycystic), and TRPML (mucolipin), grouped mainly by their homology.

12

Figure 1.2. Membrane topology of TRPs. All TRP channels contain 6 TM domains with a pore region between segments S5 and S6. The N and C termini are variable and distinguish the TRP channels. For example, TRPM channels contain an enzyme on their C termini.

13

CHAPTER 2: MEMBRANE POTENTIAL REGULATION OF TRPM7

14

Introduction

Transcellular membrane potential is critical for determining ion channel activity and

cellular signaling. It is well characterized how changes in membrane potential influence

voltage gated ion channels, and how the entry of ions, such as calcium, generates a

signaling cascade. How this potential is generated and altered by transporters and ion

channels has been fundamental to our understanding of cell and human biology.

However, how this change of electrical potential across membranes influences other

cellular processes, such as enzyme activity, has not been well described. The discovery

of a voltage sensitive enzyme phosphatase in the ascidian Ciona intestinalis sperm tail,

named Ci-VSP, in 2005 has fueled speculation of the existence of similar functioning

proteins in mammals 28. Ci-VSP consists of a transmembrane S1-S4 voltage domain

similar to that of voltage gated ion channels, along with a cytoplasmic phosphatase

domain. Its phosphatase domain acts on phosphoinositides, presumably regulating

intracellular phosophoinositide levels. Ci-VSP generates the production of

phosphatidylinositol 4,5-bisphophate PtdIns(4,5)P2 from PtdIns(3,4,6)P3. Interestingly,

several years prior to the discovery of Ci-VSP, its mammalian homologues known as

transmembrane phosphatases with tensin homology (TPTE) were identified in human

testis 29 30. However, the voltage sensing property was neither examined nor described.

Thus, the question still remains as to whether a voltage sensitive enzyme functions in

mammals, and if so, how it influences human physiology.

Phosphoinositides (PI) are widely used signaling molecules that can influence the

activity of many ion channels 31. Products of PIs, such as phosphatidylinositol 4,5-

bisphophate PtdIns(4,5)P2 and PtdIns(3,4,6)P3, comprise a minority of lipids in the inner

15

cell membrane and are highly negatively charged. Through phosphorylation at the 3,4,

and 5 sites, phosphorylated PIs can form 7 distinct molecules, and thus regulate distinct

activities. Though relative few in number at the cell membrane, these phosphorylated PIs

and its breakdown products are important signal transduction molecules. PIs are thought

to be effective signaling molecules due to its transient nature upon receptor stimulation

and formation. Such receptor stimulation is thought to activate enzymes that generate

different PIs, which proceed to modulate the activity of ion channels, transporters,

enzymes, and/or other receptors. In addition, precise localization of PIs can fine tune

where and when modulation occurs. As such, PIs have a wide array of effects on ion

channels. PIs may activate certain ion channels, while the same PI may inhibit others.

Furthermore, there has been evidence to suggest that some ion channels are completely

insensitive to the changes in PIs. Thus, insight into how and which channels are regulated

by PIs will give great insight into the inner workings of the cell.

Seminal discoveries made by Hilgemann and Ball in 1996 showed that the addition

of phospholipases reduces the activity of ion transporters and channels by decreasing

PIP2 32. The authors observed that depletion of PIP2 by phospholipase C specifically

reduced sodium-calcium exchanger activity and potassium channel activity. Addition of

exogenous PIP2 was able to restore their activities. The authors also found that the metal

aluminum may interact and interfere with the PIP2 -ion channel interaction. Since then,

many channels have been identified as PIP2 sensitive, of note which are the transient

receptor potential (TRP) channels 33.

As mentioned previously, ion channels can be activated or inactivated by the level

of a particular phosphoinositide. The canonical Drosophila TRP channel is activated by

16

DAG metabolites upon PIP2 produced by rhodopsin and Gq, though the exact mechanism

is still unclear 34 35. Channel activity is maintained in wild-type channels by continuous

calcium entry and inhibition of phospholipase C, possibly through calcium-calmodulin

inhibition, thus preserving PIP2 36. Since then, PIP2 has been shown to stimulate TRPV5,

TRPM4, TRPM5, and TRPM8, while having an inhibitory effect on the heat and capsaicin

sensitive TRPV1. Inconclusive is the effect of PIP2 on TRPM7. However, it is widely

believed that receptor activation induced PLC activation leads to PIP2 hydrolysis and

TRPM7 inactivation 37. Furthermore, it was shown that TRPM8 is regulated by PIP2, and

upon calcium entry, PLC-mediated depletion of PIP2 leads to reduced channel activity 38.

TRPM7 is a member of the melastatin subfamily of TRP channels. The first TRPM

channel was characterized in 1998 where the authors found that its expression inversely

correlated with metastatic potential 39. A few years later, TRPM7 was discovered. It is a

ubiquitously expressed, divalent permeable, cation channel that has since been

implicated in many cellular processes such as adhesion, growth, and apoptosis 40. As

mentioned before, the protein contains both channel and kinase functions, however the

role of the kinase in channel function has not been clearly elucidated 41. The channel is

permeable to both calcium and magnesium, and it is thought to regulate its respective

levels. Under physiological conditions, TRPM7 is thought to mainly carry an inward

current of divalent ions, particularly Mg2+ and Ca2+, which also act as a permeation block

to monovalent ions. The channel has constitutive activity, but its activity is increased

significantly by depletion of intracellular magnesium, whereas addition of intracellular

magnesium leads to its suppression. In addition, PLC, a known binding partner of TRPM7,

activation by Galphaq linked receptors inhibits TRPM7 activity by PIP2 depletion. It was later

17

shown that the close homologue TRPM6 is also regulated by PIP242. However, what other

endogenous stimuli reduce TRPM7 activity remains a mystery.

Located on chromosome 15 in humans, Trpm7 encodes a 1,865 amino acid

protein that is involved in activities ranging from embryonic development, ischemia,

cardiovascular disease, and cancer. Global deletion of Trpm7 leads to embryonic lethality

and targeted deletion in cells is lethal 43 44. More recently it has been shown that Trpm7

deletion leads to macrothrombocytopenia in mice and humans and platelet signaling

defects 45 46. In addition, TRPM7 dysfunction is also believed to be involved in atrial

fibrillation 47. However, the exact mechanism by which TRPM7 functions and how Trpm7

deletion may influence physiology has not been elucidated. Furthermore, how TRPM7 is

modulated by its interacting partners and the relationship between the kinase and channel

functions remains to be investigated 48. As more work is currently being done using Trpm7

knockout and mutant mice, and complemented by TRPM7 cell lines, its exact function in

physiological processes will become unveiled.

Here, we show that alterations of membrane potential may serve as one method

to modulate the activity of TRPM7. We believe the modulation occurs through changes in

PIP2. This suggests that there may be an intrinsic voltage sensing mechanism in the cell

that transduces the change in membrane potential to alter PIP2 concentration, not unlike

Ci-VSP, thus regulating TRPM7 activity.

18

Results

Hyperpolarized membrane potential inactivates the TRPM7 channel.

PIP2 has been shown to regulate TRPM7 channel activity. Furthermore, it has been

shown that an invertebrate voltage sensitive phosphatase, Ci-VSP, alters PIP2 levels,

subsequently affecting ion channel activity. Thus, we set out to determine whether such

phenomenon occurs in mammalian cells. We used human embryonic kidney cells (HEK

293) and transfected TRPM7 (also known as LTRPC7). Under whole cell configuration,

TRPM7 current increased to a maximum levels about 3 minutes following rupture, after

complete dialysis of intracellular pipette solution to chelate free intracellular Mg2+. After a

steady-state current was reached, a low voltage protocol was applied. Cells were held at

either -100mV or -120mV, with interspersed ramp protocols to monitor TRPM7 activity

(Figure 2.1A-C). TRPM7 current steadily decreased in both inward and outward

directions. In addition, we used a step protocol holding at -100mV and saw a steady

decrease in inward TRPM7 activity at -100mV (Figure 2.1D). Furthermore, to verify that

perturbations of intracellular content did not affect our results, we also used perforated

patch clamp at low voltages. We saw similar decreases in current as well (Figure 2.1E).

These results suggest that a hyperpolarized membrane potential leads to reduced

TRPM7 activity.

Dynamic PIP2 changes with membrane potential

To determine what factors may be influencing TRPM7 channel activity at hyperpolarized

membrane potentials, we first looked at PIP2, a well-described molecule involved in

TRPM7 gating. In order to monitor subtle changes in PIP2 levels at the cell membrane,

19

we used Forster resonance energy transfer (FRET) using cyan fluorescent protein (CFP)

and yellow fluorescent protein (YFP) both conjugated to a Pleckstrin homology (PH)

domain, which binds to PIP2 and is natively found in proteins such as heterotrimeric G

proteins. The ratio of YFP/CFP over time has been previously shown to effectively track

the change in levels of PIP2 on the cell surface. Thus, we transfected both CFP and YFP

proteins in a LTRCP7 cell line. Using simultaneous FRET and patch clamp

electrophysiology, we were able to track both TRPM7 activity as well as PIP2 in real time.

Similar to what we observed previously, we saw TRPM7 activity decrease at negative

holding potentials. We also saw a concurrent decrease in FRET ratio, indicating that

hyperpolarized membrane potential reduces FRET at the cell membrane, suggesting that

PIP2 levels may be modified or decreased (Figure 2.2).

Addition of exogenous PIP2 does not prevent TRPM7 inactivation

As our FRET data indicated that PIP2 levels may be declining, we sought to test whether

exogenous PIP2 might restore TRPM7 current in the setting of hyperpolarization. Thus

we added exogenous diC8- PIP2 (20uM) in the pipette solution and ran the low voltage

protocol. We could not observe any substantial restorative effects of exogenous PIP2 on

TRPM7 current using the low voltage protocol (Figure 2.3). However, as PIP2 location

and level and its interaction with TRPM7 are tightly regulated in a time dependent manner,

the exogenous PIP2 may not be sufficient to reverse the effects of low voltage.

20

Divalent ions are not likely responsible for low voltage induced change

Although our intracellular pipette solution contained EGTA for chelating divalent cations,

we wanted to further exclude the possibility of divalent interaction with the channel and/or

PIP2 in regulating the low voltage effect that we saw. As sustained hyperpolarization

drives positive charges to enter the cell, we wanted to exclude any such divalent

interactions. It has previously been shown that multivalent cations may interfere with the

PIP2 -ion channel interaction 49 50. Thus, using our LTRPC7 cell line with the low voltage

protocol, we switched the extracellular bath from normal Tyrode’s to a solution which is

divalent free (DVF). This DVF solution altered the I-V shape of LTRPC7, as expected

(Figure 2.4). However, upon stimulation with the low voltage protocol, the decrease in

both inward and outward current persisted, suggesting that divalent cations may not play

the primary role in the low voltage response (Figure 2.4).

Discussion

We report in this paper for the first time that hyperpolarization of membrane

potential leads to decreased TRPM7 ion channel activity (Figure 2.5). We show that at

low voltage there is likely to be a change of PIP2. However, the exact mechanism of how

PIP2 levels are altered remains unclear. Our FRET data indicate that PIP2 levels may be

changing. However, there may also be a change in PIP2 availability at the cell surface at

low membrane potential, thus altering FRET and potentially ion channel interaction. Thus,

there may be a similar functioning voltage sensitive enzyme in humans as that seen in

Ciona intestinalis. However, further validation is required. One mechanism explaining our

phenomenon is the presence of functional TPTE or TPIP proteins that are homologous

21

to Ciona intestinalis Ci-VSP. Another possibility is that phosphatases or kinases may be

recruited to the cell surface by interacting proteins or lipids in order to modify PIP2 levels

at the cell surface. Thus, various kinases, phosphatases, or scaffolding proteins may be

involved in this low voltage phenomenon. We have also seen that this low voltage

response occurs in other PIP2 dependent cells. Using the same low voltage protocol, we

saw decreases in Kir2.1B channel activity, suggesting that hyperpolarization may induce

PIP2 changes that affect various ion channels and thus broadly influence cellular

physiology.

PIP2 regulates a large number of ion channels and recent studies in the crystal

structure of TRP channels may yield important clues as to how PIP2 regulation occurs. In

addition, recent work has suggested that membrane potential may influence the clustering

of PIP2 at the cell membrane to influence signaling pathways 51. Furthermore, voltage

may influence the localization of phosphoinositide regulators to influence TRP channel

activity 52. Similar to the question of how low voltage induces changes, a related question

remains as to how high voltage may influence PIP2 levels. We initially started with

hyperpolarization voltage protocols in order to keep the membrane potentials at

physiological values. However, how membrane depolarization alters PIP2 availability

remains an interesting question and to be investigated. In summary, our knowledge of

how TRP channels and other ion channels may be regulated by factors that influence

PIP2 levels is of critical importance in order to determine normal cellular physiology and

the changes that occur in pathophysiological conditions.

22

Materials and Methods

Molecular biology.

LTRPC7 cell lines were obtained from Dr. A.M. Scharenberg. PH-CFP and PH-YFP

plasmids were obtained from Dr. T. Balla.

Electrophysiology.

Whole cell and perforated patch currents were recorded using an Axopatch 200B

amplifier. Data were digitized at 5 or 10kHz, and digitally filtered off-line at 1 kHz. Patch

electrodes were pulled from borosilicate glass and heat polished to form a final resistance

around 5 MΩ when filled with internal pipette solution. Series resistance was

compensated up to 90% to reduce series resistance errors.

For whole cell current recordings, voltage stimuli lasting 200 ms were delivered at 1 s or

5 s intervals, with either voltage ramps or voltage steps ranging from -120 to +100mV or

-100mV, respectively. For low voltage holding ramp protocols, cells were held at either -

100mV or -120mV before intermittent ramp tests. A quick perfusion system was used to

exchange extracellular solutions, with complete solution exchange achieved. The internal

pipette solution consisted of (in mM) 145 Cs-methanesulfonate, 8 NaCl, 10 EGTA, and

10 HEPES, with pH adjusted to 7.2 with CsOH. In some cases, BAPTA was used as a

chelator for stronger chelating effects, but effects were similar to EGTA. For PIP2

experiments, we added 20uM diC8- PIP2 to the internal pipette solution and adjusted pH

accordingly. The standard Tyrode’s extracellular solution for whole cell recording

consisted of (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 10 HEPES, and 10 glucose, with pH

adjusted to 7.4 with NaOH. For conditions using DVF perfusion, the solution contained

145 NaCl, 10 HEPES, 5 EGTA, 2 EDTA and 10 glucose with pH adjusted to 7.4.

23

MaxChelator was used to calculate free Ca2+ and Mg2+ concentrations. For perforated

patch recordings, Nystatin (200ug/mL) was added into the pipette solution and pH

adjusted accordingly.

FRET measurements.

LTRPC7 cells were plated on 35mm cell culture dishes and transfected with PH-CFP and

PH-YFP plasmids using Lipofectamine 2000 and OptiMEM. After incubation in OptiMEM

for about 5 hours, the media was replaced with 10% BGS in DMEM F12 and 2ug/mL

tetracycline for induction. The cells were incubated overnight and split with Trypsin

(0.25%) the following day onto round glass coverslips coated with poly-L-lysine for 30

minutes. Fluorescence intensities of CFP and YFP were collected at a rate of 3 Hz using

CoolSNAP HQ2 (Photometrics) and data were analyzed using NIS-Elements (Nikon). The

YFP/CFP ratio was used for determining the percent of FRET change.

Data analysis.

Pooled data are presented as mean +/- SEM. Statistical comparisons were made using

two way analysis of variance (ANOVA) and two tailed t-test with Bonferroni correction.

p<0.05 indicate statistical significance.

24

Figure 2.1A. Hyperpolarized membrane potential inactivates the TRPM7 channel. TRPM7 overexpression in HEK 293 cells leads to robust current under patch clamp

electrophysiology in which intracellular divalent cations are buffered. The typical

outwardly rectifying current is observed using a ramp protocol. The voltage protocol can

be modified as shown (bottom), where the cell can be held at high or low voltages after

each sweep. Low voltage holding leads to reduced TRPM7 current (red). The current can

be recovered by switching back to 0mV holding.

-100 -50 50 100

100

200

300

mV

pA/pF

Recovery

Initial-120

25

Figure 2.1B. TRPM7 inactivation shows time dependent inactivation and recovery. TRPM7 overexpression in HEK 293 cells leads to robust current under patch clamp

electrophysiology in which intracellular divalent cations are buffered. Both max inward

(red) and outward (black) currents are plotted against time (in seconds). As the pipette

solution dialyzes into the cell, max TRPM7 current is reached. Upon holding the cell at -

120mV, both inward and outward current quickly reduce. The current recovers soon

following holding potential at 0mV.

100 200 300 4000

100

200

300

Initial -120 recovery

Time (sec)

pA/p

F

26

Figure 2.1C. Recovery after inactivation approaches near maximum. TRPM7

overexpression in HEK 293 cells leads to robust current under patch clamp

electrophysiology in which intracellular divalent cations are buffered. The outward current

is shown at the end of the respective stimulus (initial, -120, and recovery). All of the cells

tested show a decrease in current at -120mV, and are able to recover near maximum

current.

Initial -120 Recovery0

100

200

300

400

500

pA/pF

27

Figure 2.1D. Hyperpolarized membrane potential by step protocol also inactivates the TRPM7 channel. TRPM7 inward current was assessed over a period of 20 seconds

by using the step protocol. Under this protocol, a continuous voltage of -100mV was

applied and current was simultaneously recorded. As shown, the current decreases over

time, similar to what we observed using the ramp protocol in Figure 2.1B.

10 20

-20

-15

-10

-5

0

Time (sec)

pA/p

F

28

Figure 2.1E. Preservation of intracellular contents by perforated patch technique recapitulates whole cell results. Perforated patch clamp technique was used to

minimize the dilution of endogenous intracellular components. Nystatin (200ug/mL) was

added to the intracellular pipette and current stabilization was observed. Following

stabilization, low voltage was applied.

-100 -50 50 100-200

200

400

600

mV

pA

Initial-100

29

cell background0.00

0.05

0.10

0.15

0.20

Rat

io d

ecre

ase

at L

V

Initial -1200

200

400

600

pA/pF

a = 0mV b = -120mV c = 0mV

30

Figure 2.2. Dynamic PIP2 changes with membrane potential. Changes of PIP2 were

measured using FRET with PH-CFP and PH-YFP. The change in YFP/CFP ratio was

traced over time at low voltage and recovery holding potentials, while simultaneously

measuring current activity. Only the outer membrane region of the cell was included in

the ratio measurements, as this area gives the changes in membrane bound PIP2 (top).

Upon low voltage protocol, FRET decreased rapidly (middle), which also parallel a

decrease in TRPM7 current (lower left). The FRET ratio decreased about 15% at low

voltage relative to 0mV, whereas the background FRET ratio remained steady (lower

right).

31

Figure 2.3. Addition of exogenous PIP2 does not prevent TRPM7 inactivation. Exogenous PIP2 (diC8- PIP2, 20uM) was included in the intracellular pipette solution.

Addition of PIP2 did not lead to lower inactivation following low voltage protocol (top).

Additionally, the kinetics of inactivation were not changed when PIP2 was included in

the pipette solution (bottom).

w/ PIP2

w/o PIP2

0.0

0.1

0.2

0.3

0.4

0.5

frac

tion

decr

ease

at -

100m

V

w/ PIP2

w/o PIP2

0

50

100

150

time

to m

ax d

ecre

ase

(sec

)

32

Figure 2.4. Divalent ions are not likely responsible for low voltage induced change. Tyrode’s extracellular solution was replaced with a divalent free (DVF) solution

to eliminate the role of divalent ions in mediating TRPM7 inactivation. DVF solution was

perfused and once a steady current was maintained (black), low voltage was applied

(red). Low voltage was still able to inactivate TRPM7 in DVF conditions.

-100 -50 50 100

-300

-150

150

300

mV

pA/pF

Initial-100

33

Figure 2.5. Model of TRPM7 inactivation by low voltage. PIP2 is generated/degraded

by a number of physiological processes. Which, if any, pathway is responsible for the

voltage inactivation remains to be seen. TRPM7 is regulated by a variety of stimuli

(bottom). Low voltage may also influence one of these pathways, such as PLC

activation, to lead to TRPM7 inactivation.

34

CHAPTER 3: TRPM2 MODULATION OF INFLAMMATION AND CARDIOVASCULAR DISEASE

35

Introduction

Changes that occur in heart disease are not limited to the heart, but throughout the

individual. Individuals with coronary artery disease (CAD) and subsequent myocardial

infarction (MI) are susceptible to multiple other vascular-related conditions such as stroke,

peripheral vascular disease, and ischemic kidney disease, the common underlying

connection being atherosclerosis. In patients, atherosclerosis has been associated with

high levels of the inflammatory cytokine IL-6 and the biomarker C-reactive protein along

with an increase in inflammatory peripheral monocytes 53 54. Apoe-ko mice (in combination

with a high fat diet), which lack a protein involved in hepatic uptake of circulating

cholesterol, enable atherosclerosis studies in mice 55. In mouse models of

atherosclerosis, systemic changes such as a selective expansion of circulating

inflammatory monocytes are seen. Moreover, monocytes have been shown to be involved

in MI recovery at both acute and chronic phases of infarct healing 18. Initial recruitment of

inflammatory Ly6Chi monocytes is followed by recruitment of reparative Ly6Clo monocytes

in the damaged heart, a sequence that is necessary to ensure proper healing. A recent

study showed that atherosclerosis induces a chronic elevation of inflammatory Ly6Chi

monocytes which impairs infarct healing. Furthermore, myocardial infarction has been

shown to accelerate atherosclerosis and increase the chances for a subsequent MI. Thus

a better understanding of the role of monocytes in MI using clinically relevant models of

atherosclerosis is critical. Loss of regulation of inflammatory monocytes can propagate

harmful cardiac remodeling and lead to alterations in chamber size and contractility,

ultimately leading to sudden death or recurrence, arrhythmia, heart failure, and stroke.

36

To dissect the mechanism of how inflammatory monocytes contribute to I-R injury,

we focus on TRPM2, an oxidative stress activated Ca2+-permeable ion channel 56. TRPM2

is expressed in the brain, endothelial cells, heart, and immune cells, such as

monocytes/MΦs. TRPM2 is activated through an intracellular domain that bind Ca2+ and

oxidative stress products such as hydrogen peroxide, both of which are increased in heart

disease. Moreover, TRPM2 activation by reactive oxygen species in immune cells has

been thought to play a crucial role in inflammatory diseases. Although the precise

physiological function of TRPM2 is unknown, a study has shown that TRPM2 activation

and subsequent Ca2+ entry in monocytes aggravates chemically induced colitis by

production of the paracrine signal CXCL8 which leads to an aggravation of inflammation

57. Another study has implicated neutrophil TRPM2 in exacerbating cardiac injury after

ischemia-reperfusion (I-R) injury by increased neutrophil adhesion 58. Finally, a recent

finding suggests that TRPM2-mediated Ca2+ entry is necessary in MΦs for NLRP3-

inflammasome activation and secretion of the pro-inflammatory molecule IL-1β 59.

Here we study atherosclerosis, as well as MI in a background of atherosclerosis to

closely parallel human disease, where MI is not an isolated event, but occurs in the

presence of vascular disease and inflammation. We thus overcome a barrier in

cardiovascular research which lacks such studies, where alterations in inflammatory

status, cytokine profile, and leukocyte populations abound. As survival rates after the

initial episode of MI are increasing due to improving acute interventions after MI, there is

an increasing need to develop treatments to halt the progression of heart disease. This

research takes the first steps into gaining new insight into how modulation of an ion

channel, TRPM2, after I-R injury can harness the protective aspects of inflammation,

37

while reducing its deleterious consequences. Additionally, as TRPM2 has been linked to

the inflammasome pathway, TRPM2 may serve as an effective target for other

inflammatory diseases. As there has been a push toward developing inhibitors of NLRP3

inflammasome and IL-1β for inflammatory and fibrotic diseases, the potential therapy

through TRPM2 holds promise.

Results

Trpm2 deletion protects against atherosclerosis

As TRPM2 is involved in various inflammatory conditions, we wanted to test whether

TRPM2 influences atherosclerosis disease progression. Using our mouse model of

atherosclerosis, we saw that Apoe knockout mice develop substantial aortic

atherosclerosis following treatment with a high fat diet (HFD), as measured by Oil Red

staining of the aorta. However, we were very surprised to see that the addition of global

Trpm2 knockout led to a reduction in large vessel atherosclerosis as compared to Apoe

knockout alone (Figure 3.1).

Trpm2 deletion protects against myocardial infarction

Furthermore, we wanted to see test how TRPM2 mediates other forms of cardiovascular

disease. We wanted to mimic the human scenario of MI, whether there is abundant

systemic inflammation and atherosclerosis. Thus, using the mice we used above for our

atherosclerosis studies, we induced myocardial infarction using the well-established

model of myocardial infarction. At one month following MI, we saw improved heart

function in the Trpm2 knockout mice as measured by ejection fraction. Furthermore, upon

Pressure-volume loop analysis at two months, there were increased cardiac output and

38

stroke volume, among other parameters, in the Trpm2 knockout group compared to wild-

type (Figure 3.2). This suggested that TRPM2 may be contributing to the deleterious

effects after MI, presumably by reducing inflammation.

Systemic changes in circulating myeloid cells during atherosclerosis and MI

Previous reports have shown that both atherosclerosis and MI contribute to the increased

levels of circulating inflammatory monocytes. Thus, we wanted to determine how TRPM2

influences this population by flow cytometry, which may be contributing to the

atherosclerosis and MI phenotypes we observed (Figure 3.3A). Trpm2 knockout did not

affect basal myeloid cell populations (Figure 3.3B). Flow cytometry of peripheral blood

obtained from mice treated with HFD over a long period of time showed increased levels

of circulating myeloid cells, predominantly due to the increased circulating neutrophil

population. However, Trpm2 knockout mice showed reduced neutrophil burden, more

similar to mice without atherosclerosis (Figure 3.3C). However, following myocardial

infarction, we were not able to observe a difference in circulating inflammatory Ly6Chi

cells in Trpm2 knockout mice (Figure 3.3D). Our results suggest that our method may not

have the sensitivity to detect whether TRPM2 may be influencing the circulating

peripheral blood cell population after myocardial infarction, and/or that the main

contributor effect of TRPM2 is through the change in circulating neutrophil population.

However, we do see TRPM2 expression in bone marrow progenitor cells, suggesting a

possible role in the bone marrow reserve (Figure 3.4).

39

Discussion

Our results show for that deletion of Trpm2 is protective against cardiovascular

disease (Figure 3.5). We show that global Trpm2 knockout reduces atherosclerotic

burden. Furthermore, Trpm2 knockout preserves heart function after a model of

myocardial infarction in a setting of concurrent atherosclerosis. We have also created cell

type specific knockouts of Trpm2 in the endothelium (Cdh5) and myeloid lineage (Cx3cr1

and Cd11b) in order to determine the predominant cell type involved in this process

(Figure 3.6).

Preliminary results suggest that myeloid cells are involved in the process. Flow

cytometry data indicate that circulating neutrophils are increased in atherosclerosis,

though we were not able to observe any significant differences in circulating inflammatory

monocytes after myocardial infarction. The neutrophil levels are reduced in Trpm2

knockout mice during long term atherosclerosis, suggesting that TRPM2 coordinates the

myeloid population in these disease processes. To be determined is how exactly TRPM2

mediates such deleterious effects.

One possible mechanism in which TRPM2 is involved is through changes in

cellular adhesion and/or secretion of chemokines. We believe that the pro-inflammatory

cytokine IL-1β may contribute to the inflammatory processes observed. However, other

studies have shown that TRPM2 is involved in the secretion of other chemokines such as

CXCL2, which acts to recruit other inflammatory cells. Furthermore, as circulating myeloid

levels are changed in Trpm2 knockout mice, another possible role for TRPM2 is through

its role in the release of inflammatory cells from myeloid reservoirs such as the spleen

and bone marrow. As TRPM2 has been shown to be expressed in the autonomic nervous

40

system, it may be responsible in mediating the release through actions on the sympathetic

or parasympathetic pathways 60.

Materials and Methods

Mice.

Global Trpm2-ko mice obtained from Y. Mori were used which carry a deletion in the exon

encoding for transmembrane segment 5. For atherosclerosis experiments, Apoe

knockout mice were used obtained from Jackson laboratories. All mice were in the

C57BL/6 background. Apoe-ko are referred to as “SKO” (single knockout) and Apoe-ko

with Trpm2 deletion are referred to as “DKO” (double knockout). High fat diet (HFD) food

(21% fat, 0.15% cholesterol by weight obtained from Harlan) was fed starting at 5 weeks

of age to generate atherosclerosis.

Echocardiography and PV loop.

Echocardiography heart function measurements were performed by probing the left

ventricular mid region using Vevo 770 in two-dimensional M mode. PV loop

measurements were performed using Scisense Pressure-Volume Measurement system

with catheter (Transonic) and ADVantage PV system (Scisense) and analyzed with

LabScribe (iWorx). Mice were anesthetized using 3% isoflurane.

Atherosclerosis staining.

Quantification of plaque burden was performed following HFD treatment. Aorta were

harvested from mice and stained using standard Oil Red O staining procedures.

Myocardial infarction model.

41

LAD ligation was used to produce I-R injury. This procedure followed standard techniques

of 45 minute ischemia following by reperfusion 61.

Flow cytometry.

Peripheral blood was collected at various time points using tail vein bleed. After 10-12

drops of blood were collected, leukocytes were separated and stained for flow cytometry.

Alexa Fluor 700 Rat Anti-Mouse Ly-6C (BD), FITC antimouse Ly-6G (BioLegend), Biotin

anti mouse F4/80 (BioLegend), PE CF594 streptavidin (BD), Anti-mouse CD3e PE-

cyanine 7 (eBioscience), Anti-human/mouse CD45R (B220) PE-Cyanine 7 (eBioscience),

anti-mouse NK1.1 PE-Cyanine 7 (eBioscience), Anti-mouse CD11b eFlour 450

(eBioscience), APC anti-mouse Cx3cr1 (Biolegend) were used.

Data analysis.

Pooled data are presented as mean +/- SEM. Statistical comparisons were made using



42

Figure 3.1. Trpm2 deletion reduces atherosclerosis. Mice fed a HFD develop

atherosclerosis throughout the aorta, as measured by Oil Red O staining. The deletion of

Trpm2 leads to a significant protection in atherosclerosis levels as indicated by the

representative image (left) and by the reduced lesion area. Lesion area was quantified as

area of red stained by Oil Red O vs. the total surface area of the aorta (right).

43

44

Figure 3.2. Trpm2 deletion protects against myocardial infarction. Deletion of Trpm2

was previously shown in our lab to protect against detrimental cardiac remodeling (top

left). Using echocardiography (top right) and PV loop analysis, we assessed how effective

Trpm2 deletion is in protecting against myocardial infarction in a model mimicking human

disease. Trpm2-ko mice in the Apoe knockout background (DKO) had improved heart

function as measured by echocardiography and PV loop analysis 3 weeks post I-R injury

(bottom).

45

Figure 3.3A. Flow cytometry process. Peripheral blood was collected by tail vein bleed

from mice at various time points. The leukocytes were isolated and labeled with markers

to exclude lymphocytes. Myeloid cells were gated for and these cells were further

subdivided in neutrophils and Ly6C high or low monocytes.

46

Figure 3.3B. Trpm2 deletion does not alter myeloid subpopulations in the healthy state. Global Trpm2 knockout, Cd11b specific knockout, and WT mice peripheral blood

cells were isolated from mice in the healthy state (with no atherosclerosis). Under these

conditions, there were no significant differences among the three groups in terms of the

basal myeloid cell populations (along with the neutrophil and Ly6Chi populations).

47

Figure 3.3C. Systemic changes in circulating myeloid cells during atherosclerosis. Peripheral blood cells obtained from SKO mice treated with HFD showed an increase in

myeloid population, mainly due to the increase in neutrophil population. This increase

was abrogated in DKO mice.

48

Figure 3.3D. Myocardial infarction does not alter peripheral myeloid populations. Peripheral blood was collected at various time points before and after myocardial

infarction in SKO and DKO mice fed with HFD. PreLAD and at 1 day and 1 week following

LAD, there were no significant differences in overall myeloid cells, Ly6Chi and Ly6Clo cells

between SKO and DKO mice. Later time points also showed no differences (data not

shown).

49

Figure 3.4. TRPM2 is expressed in bone marrow progenitor cells. Progenitor cells

from the bone marrow were collected and sorted by flow cytometry into common myeloid

progenitor (CMP) and LKS cells. These cells were then analyzed for TRPM2 expression

by patch clamp electrophysiology.

TRPM2 characterization in lineages

B cells

T cells

Monocytes CMP

LKS0

100

200

300

400

pA

50

Figure 3.5. Model of TRPM2 induced inflammation in myeloid cells. Oxidative stress

activates TRPM2, which mediates calcium entry into myeloid cells. This leads to

activation of downstream signaling pathways such as the inflammasome pathway,

leading to the secretion of IL-1β and propagation of inflammation. Targeting TRPM2 may

serve to reduce inflammation and its consequences such as atherosclerosis.

51

Figure 3.6. Myeloid specific, inducible deletion of Trpm2 to study atherosclerosis and myocardial infarction. We have generated stable mouse lines that allow Trpm2

deletion at a specific time point only in myeloid cells. We currently have Cx3cr1 Cre mice

that are tamoxifen inducible with Trpm2 fl/fl and in the Apoe knockout background.

52

CHAPTER 4: TARGETING TRPM7 TO REVERSE CARDIAC FIBROSIS

53

Introduction

Cardiac fibrosis is a key component in many heart diseases including cardiac

hypertrophy, heart failure, and arrhythmia. Fibrotic depositions are laid in response to

acute or chronic stressors, and are generally thought to be a protective mechanism.

However, over time, the remodeled tissue can serve as substrate for atrial fibrillation

and/or lead to reduced heart function over time due to a vicious cycle of extracellular

deposition leading to decreased contractility. Despite intensive efforts to find ways to treat

cardiac fibrosis, there has been very little progress.

The main cell type involved in the deposition of extracellular matrix in the heart is

the cardiac fibroblast (cFB) 62. Often neglected in the field of cardiac research, these

fibroblasts play a critical role in cardiac physiology and pathophysiology, with some

literature suggesting its involvement in setting the resting membrane potential and

influencing excitability. It is also believe that these fibroblasts may be both electrically and

physically coupled to cardiomyocytes. Recent studies have shown cFBs as the cell type

involved in TGFβ-induced fibrosis. Thus, targeting cFBs to reduce fibrosis has been a

topic of recent interest.

One potential target for fibrosis is through TRPM7. TRPM7, a “chanzyme” due to

its dual characteristics of being a channel while also containing a kinase domain, has

been implicated in many different types of fibrosis, ranging from liver to pulmonary to

more recently, cardiac fibrosis 63. Previous research in our lab has shown TRPM7 to be

upregulated in patients with atrial fibrillation. Furthermore, TRPM7 was shown to be

involved in the differentiation of cardiac fibroblasts into myofibroblasts following TGF-β

stimulation 64.

54

Here we show that deletion of Trpm7 in a model of hypertensive heart disease

protects against negative cardiac remodeling. In addition, using cell type specific Trpm7

knockout, we show that Trpm7 knockout specifically in cardiac fibroblasts may be critical

in providing this protective effect. Our results suggest that TRPM7 may be a novel target

for reducing cardiac fibrosis, and potentially fibrosis in other tissues as well.

Results

Deletion of Trpm7 attenuates fibrosis and improves heart function

We sought to determine if TRPM7 is involved in cardiac fibrosis. Using the

transverse aortic constriction model (TAC), we investigated the role of Trpm7 deletion.

As Trpm7 deletion in development is lethal, we used tamoxifen inducible global Trpm7

deletion. As soon as 1 week after TAC, we observed significant protection by Trpm7

deletion compared to wild type. The ejection fraction was improved, as measured by

echocardiography, up to 2 month post TAC, along with an improvement in overall survival.

Furthermore, cardiac fibroblasts isolated from TAC mice showed increased TRPM7

activity by patch clamp electrophysiology as compared to sham mice, suggesting that

TRPM7 is likely involved in negative remodeling of the heart (Figure 4.1).

Fibroblast specific deletion of Trpm7 recapitulates protective effects

To validate that TRPM7 in fibroblasts plays a key role in mediating fibrosis, we

then went ahead to use fibroblast specific Trpm7 knockout using periostin Cre and

compared the effects after TAC with myocyte specific knockout. Interestingly, we found

that Trpm7-MY-ko did not produce any protective effect in the TAC model, but Trpm7-FB-

55

ko produced similar protection as we observed using global knockout (Figure 4.2). Thus,

our results suggest that fibroblasts play a key role in mediating fibrosis through TRPM7.

Discussion

Our results show that fibroblast specific TRPM7 is involved in mediating cardiac

fibrosis. Deletion of Trpm7 produced significant protection in a pressure

overload/hypertension model. As the burden of cardiac disease and fibrosis is growing,

novel ways to reduce morbidity and mortality will need to be discovered. Here we show

that TRPM7 may serve as a potential candidate to improve heart function. However, as

TRPM7 is ubiquitously expressed, and critical for development, targeted strategies to

reduce TRPM7 activity will need to be determined.

Furthermore, the mechanism as to how TRPM7 mediates cardiac fibrosis has yet

to be determined. As TRPM7 is both a channel and kinase, one or both components may

be involved in mediating cardiac fibrosis (Figure 4.3). Future studies using channel dead

or kinase inactive forms of TRPM7 will be informative. Our lab has also shown

(unpublished data) that CaMKII may play a role in transducing the TRPM7 mediated

calcium signal during fibrogenesis, and that the TGFβ signaling pathway may also be

involved as well. Future studies to clearly elucidate the mechanism may yield great insight

into understanding fibrosis mediated by TRPM7 not only in the heart, but in other tissues

as well.

56

Methods

Mice.

Global Cre for Trpm7 knockout was induced by tamoxifen and confirmed by genotyping,

western blot and patch clamp. Control mice were the Cre- littermates. Myh6 Cre were

obtained from D. Clapham and Perisotin Cre were obtained from Dr. S.J. Conway.

Transverse aortic constriction (TAC).

A model of pressure overload was created using the established transverse aortic

constriction model 65. Briefly, a suture was place in the transverse aorta between the

brachiocephalic and left common carotid arteries. Successful constriction was measured

by Doppler before and immediately after the stricture following the procedure.

Echocardiography.

Echocardiography heart function measurements were performed by probing the left

ventricular mid region using Vevo 770 in two-dimensional M mode. Mice were

anesthetized using 3% isoflurane.

Data analysis. Pooled data are presented as mean +/- SEM. Statistical comparisons were made using



57

Figure 4.1. Deletion of Trpm7 attenuates fibrosis and improves heart function. Deletion of Trpm7 was verified using PCR, western blot, and patch clamp

electrophysiology (top left). Following TAC, Trpm7 ko mice had increased survival over 8

weeks (bottom left). In addition, heart function parameters measured by

echocardiography showed improved function in Trpm7 ko mice at all time points

measured after TAC.

58

Figure 4.2. Fibroblast specific deletion of Trpm7 recapitulates protective effects. Both myocyte and fibroblast specific knockout of Trpm7 were used in the TAC model to

narrow down the cell type responsible for mediating the protective effect of Trpm7

knockout. Although myocyte knockout did not yield any protective benefit in heart function

over WT as measured by echocardiography, the results from fibroblast specific knockout

were similar to global Trpm7 knockout. Furthermore, the HW/BW ratio and fibrosis area

were reduced in fibroblast Trpm7 knockout mice. Finally, PV loop analysis showed

improved heart function in fibroblast Trpm7 knockout mice compared to WT after TAC.

59

Figure 4.3. Schematic of TRPM7, the chanzyme. How TRPM7 mediates fibrosis in our

hypertensive model remains to be seen. The protein has both channel and enzyme

functions. Whether the function of one influences the other is still under investigation.

Calcium is an important secondary messenger as well. Our lab has also shown that

CaMKII may play a role in transducing the TRPM7 mediated calcium signal during

fibrogenesis, and that the TGFβ signaling pathway may also be involved as well.

60

CHAPTER 5: pH REGULATION OF THE GATEKEEPER, ORAI

61

Introduction

Calcium signaling is crucial in a variety of physiological and pathophysiological

processes, and thus its regulation is of critical importance. It is known that calcium release

activated calcium channel currents (ICRAC) are altered by conditions such as acidosis and

alkalization. Physiologically, intracellular alkalization is associated with membrane

depolarization, cell proliferation, and cellular differentiation, while intracellular acidosis

has been shown to be involved in apoptosis 66 67 68. Extracellular acidosis, which occurs

in settings such as myocardial ischemia, has been shown to inhibit ion channel activities

69 70. Thus, the question remains as to how pH is sensed by ion channels to induce

changes in the cell.

Similar to other channels, Icrac is inhibited and potentiated by acidic and basic pH,

respectively. ICRAC is composed of the Orai and STIM subunits, which act as the pore

forming and calcium sensing units respectively 71 72. Thus, pH may influence how these

two subunits bind, or may directly influence the activities of these proteins.

Here we show that acidosis and alkalosis alter Orai1/STIM channel functions. We

generated candidate mutations on transmembrane domain 3 and found that E106 is

responsible for pHo sensitivity when calcium is the permeant ion. We also identified E190

of Orai1 as the major sensor of pHo when sodium is the permeant ion. In addition, we

found H155 on the intracellular loop is responsible for sensing intracellular pH. Overall,

we show that these residues play a critical role in sensing pH to mediate acid/base

induced changes in channel activity.

62

Results

Effects of extracellular pH on Orai1/STIM1 currents

Orai1/STIM1 currents were recorded by inducing store depletion through EGTA.

The cells were perfused with extracellular DVF solutions adjusted to various pHs.

Orail1/STIM1 currents were significantly increased when the cell was exposed to high

pHo. On the other hand, alkaline pHo inhibited current amplitude (Figure 5.1). As shown

in Figure 5.1, the effects of pH were reversible. Furthermore, we tested the effects of pH

using Tyrode’s solution. Figure 5.1 shows that acidic pHo inhibited whereas basic pHo

enhanced Orai1/STIM1 current.

Effects of external pH on Orai2/STIM and Orai3/STIM

We also tested whether other Orai2 and Orai3 were similarly affected by pH. As

shown in Figure 5.2, both Orai2 and Orai3 currents were potentiated by external basic

pHo and inhibited by acidic pHo. These results indicated that all isoforms are capable of

responding to external pH cues, and that the pH sensor may reside in an Orai residue.

Mechanism of extracellular pH regulation

We first tested whether external protons directly regulated Orai1/STIM1. We

generated candidate mutations by neutralizing a series of negatively charged residues

along the external site of the channel or channel pore, in addition to E106D and E190D.

Interestingly the mutant E190D displayed smaller inhibition at acidic pHo and significantly

reduced potentiation at basic pHo compared to wild type when the cells were perfused

with DVF. Furthermore, the dose-response curve of E190D was shifted to the left,

indicating that E190 is essential for sensing extracellular pH sensitivity (Figure 5.3).

Effects of internal pH on Orai1/STIM1

63

We also tested how intracellular pH influence Orai1/STIM1 currents. We adjusted

intracellular pipette conditions to various acidic and basic pH, followed by measurement

of current amplitude. Similar to what we observed with external pH, we saw that acidic

internal pH reduced current amplitude as measured by patch clamp electrophysiology,

whereas basic pH potentiated current amplitude (Figure 5.4).

Molecular mechanisms of internal pH sensitivity of Orai1/STIM1

To identify the residues responsible for sensing internal pH, we generated a series

of mutations at candidate pH sensing residues including histidine, glutamic acid, and

cysteine. One of our candidates, H155F, showed poor response to both acidic and basic

internal pH, suggesting that this residue on Orai1 may be responsible to sensing

intracellular pH (Figure 5.5).

Discussion

Here we show that Orai/STIM1 channels involved in ICRAC are regulated by both

internal and external pH. Furthermore, we identify the residues responsible for detecting

both intracellular and extracellular pH sensitivity. In addition, the residues involved in

detecting extracellular pH vary by the type of permeant cation. We tested how

extracellular Tyrode’s solution with calcium as the permeant ion may alter pH sensitivity.

We found that the mutant E190D had similar pH sensitivity to wild type, but the mutant

E106D lost sensitivity (Figure 5.6). This suggests that E106 is the major pH sensor when

calcium is the major permeant ion. As pH fluctuates during physiological and

pathophysiological processes, how pH is sensed and how it then influences channel

activity and calcium dynamics will yield important insight into tackling diseases in which

pH changes occur 73.

64

Methods

Molecular biology.

The constructs Orai1-3 and STIM1 were purchased from Addgene. Mutations of Orai1

were performed using site directed mutagenesis (QuickChange).

Cell culture.

HEK 293 cells were grown in DMEM/F12 media supplemented with 10% FBS and

100U/ml penicillin and 100mg/ml streptomycin. The cells were incubated in a 37°C

humidity controlled incubator at 5% CO2. Plasmids were transfected using Lipofectamine

2000.

Electrophysiology.

Whole-cell currents were recorded using an Axopatch 200B amplifier. Data were digitized

at 10 or 20kHz and digitally filtered offline at 1kHz. Borosilicate glass electrodes that were

heat polished to a resistance of 5-7 MΩ were used. Series resistance was compensated

up to 90% to reduce series resistance errors. For whole cell recordings, voltage stimuli

lasting 200 ms were delivered at 1s intervals with voltage ramps ranging from -120 to

+100mV. A holding potential of 0 mV was used. The perfusion and vacuum system

allowed efficient exchange of extracellular solution.

The internal pipette solution consisted of (in mM) 145 Cs-methanesulfonate, 8 NaCl, 10

EGTA and 20 HEPES, with pH adjusted to 7.2 with CsOH. MgCl2 (3mM) was included in

the pipette solution to block any endogenous current. pH was adjusted using citrate acid

and CsOH.

Extracellular solution consisted of divalent-free solution (DVF) containing (in mM) 145 Na

SO3CH3, 20 HEPES, 5 EGTA, 2 EDTA and 10 glucose. Tyrode’s solution contained (in

65

mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose. HCl was used to

adjust to acidic pH and NMDG was used to adjust to basic pH.

Data analysis.

Pooled data are presented in mean +/- SEM. Dose–response curves were fitted by an

equation of the form: E= Emax (1/[1+(EC50/C)n]), where E is the effect at concentration C,

Emax is maximal effect, EC50 is the concentration for half-maximal effect and n is the Hill

coefficient. Statistical comparisons were made using analysis of variance (ANOVA) and

two tailed t-test with Bonferroni correction. p<0.05 indicates statistical significance.

66

Figure 5.1. Acidic and basic external pH on Orai1/STIM1 channels. Orai1 and STIM1

were overexpressed in HEK 293 cells and current was detected by patch clamp

electrophysiology using a ramp protocol. Extracellular acidic pH inhibited while basic pH

potentiated current (left). The inward current at -100mV showed a dose dependent

response (right).

67

Figure 5.2. Effects of pH on Orai2 and Orai3. We also tested Orai2 and Orai3 at different

extracellular pH. Similar to the results observed with Orai1, we saw inhibition at low pH

and potentiation at high pH.

68

Figure 5.3. pH effects on Orai1 mutants. To test which residue is responsible for

extracellular pH sensing, we generated candidate mutations by neutralizing a series of

negatively charged residues along the external site of the channel or channel pore. The

mutant E190D displayed reduced pH dependence compared to wild type

69

Figure 5.4. Effects of internal pH on Orai1/STIM1. Similar to the above protocol, we

used a ramp protocol to elicit current and adjusted the intracellular pipette solution to

various pH. Similar to extracelllar pH, acidic internal pH inhibited and basic internal pH

potentiated current.

70

Figure 5.5. H155 as the intracellular pH sensor of Orai1/STIM1. H155F produced

diminished responses to acidic and basic internal pH (top right), suggesting that H155 is

responsible for sensing intracellular pH. Furthermore, the dose response curves of H155F

were showed reduced response to pH.

71

Figure 5.6. Effects of external calcium on pH sensitivity of Orai1/STIM1. When

calcium is included in the extracellular solution (in this case, 20mM to enhance current

level), we saw that E106 was the residue responsible for pH sensing, whereas the other

residues including E190D showed normal response.

72

CHAPTER 6:

CONCLUSIONS, DISCUSSION, AND THE FUTURE TR(I)PS

73

Since its initial discovery in Drosophila, TRP channels have become to be viewed

as versatile ion channels with diverse functions. Conserved throughout species and

present ubiquitously in almost every cell type, TRP channels mediate activities ranging

from sensation to development to cancer. These unique channels have distinct N and C

termini that confer the ability to respond to many different stimuli. None of the channels

in the family are more unique than TRPM7. As a bifunctional channel enzyme, the protein

serves as both an ion channel and an enzyme. However, the exact nature of how the ion

channel and enzyme are related in function has yet to be fully uncovered. Here we have

shown this unique channel-enzyme can be regulated by membrane potential. Specifically,

low voltage has the ability to reduce TRPM7 activity. Our results suggest that this effect

is due to a depletion in level or availability of PIP2 to TRPM7. This leads us to believe that

there may be a voltage sensitive enzyme not dissimilar to that of Ci-VSP that may be

regulating PIP2 levels at different voltages. What the specific mechanism of voltage

sensing in mammalian cells is will be exciting to see, and an avenue that our lab will

continue to pursue.

Furthermore, TRP channels have recently been identified as important players in

cardiovascular disease. TRPM2 is a well-known oxidative stress sensitive ion channel

that has been thought to mediate intercellular communication through the release of

cytokines and chemokines. We have shown that TRPM2 is involved in the atherosclerotic

disease process, as deletion of Trpm2 is protective in mouse atherosclerosis models. We

observe that mice with long term atherosclerosis have increased levels of circulating

neutrophils, and this is reversed in Trpm2 knockout mice. This highly suggests that

TRPM2 plays an essential role in mediating myeloid and neutrophil cells, though the exact

74

mechanism has not been elucidated. We have generated specific knockouts of Trpm2 in

various tissues such as the myeloid cells and endothelial cells to help us answer this

important question.

Finally, we show one of the roles of TRPM7 as a mediator of cardiac fibrosis. Our

lab has previously shown the role of TRPM7 in the differentiation of cardiac fibroblasts to

myofibroblasts. Here we show that TRPM7 in vivo can mediate fibrosis in the setting of

hypertensive heart failure. We see that deletion of Trpm7 can have a protective effect on

the heart as early as one week after induction of hypertension, which is maintained

throughout the life of the animal. Furthermore, the function of TRPM7 is primarily in the

cardiac fibroblast, as fibroblast specific knockout recapitulates the protective effects

observed in global Trpm7 knockout. The next question we will tackle is how TRPM7

mediates fibrosis and whether the chanzyme properties of the protein are utilized if at all

in this process.

Mainly considered channels important in sensory perception, the potential roles of

TRP channels in physiology and pathophysiology are now thought to be limitless. As

complex ion channels that are ubiquitous and evolutionarily conserved, we are beginning

to appreciate more and more the diverse biological roles of these channels. Similarly, as

parallel studies investigate how these channels are endogenously regulated, more insight

will be gained into their biological roles. Recent papers that have published the structure

of various TRP channels. The understanding of structural conformation will also prove to

be invaluable in the understanding of the physiological and pathophysiological roles of

TRP channels not only in cardiovascular disease but also in other diseases.

75

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TRP Channels in Cardiovascular Disease