Autoimmunoreactive IgGs Against Cardiac Lipid Raft-Associated Proteins in Patients with POTS

Abstract

Lipid rafts are specialized plasma membrane microdomains that serve as platforms for integrating cellular signal transductions. We have recently reported that autoantibodies against cardiac membrane proteins are present in patients with postural orthostatic tachycardia syndrome (POTS). In this study, we examined the presence of autoimmunoreactive IgGs against lipid raft proteins in these patients. IgGs were purified from the sera of 10 patients and 7 normal controls. Cardiac lipid raft preparations were isolated from normal human heart tissue. The lipid raft-associated proteins were resolved by 2DE and immunoblotted against IgGs from each subject. Protein spots that reacted specifically with patient IgGs were identified by nanoLC-MS/MS. Thirty-four such protein spots, and 72 unique proteins were identified. The targets of autoimmunoreactive IgGs include proteins associated with caveolae structure, adrenergic signaling, calcium signaling, cytostructures, chaperone and energy metabolism. Multiple pathways were involved including those that regulate caveolae-mediated signaling, oxidative phosphorylation, fatty acid metabolism, protein ubiquitination, and cardiac β-adrenergic signaling. Our results suggest that cardiac lipid raft-associated proteins are targets of autoimmunoreactive IgGs from patients with POTS. Autoimmunity may play a role in the pathogenesis of POTS.

Postural orthostatic tachycardia syndrome (POTS) is a common form of orthostatic intolerance, and the etiology of the syndrome is complex and unexplained in most patients. Various causes and mechanisms have been proposed for the disease including autoimmunity and ganglionic acetylcholine receptor (AChR) antibodies¹. We have recently reported the presence of autoantibodies against cardiac membranes in POTS patients². In this study, we examined whether cardiac proteins in lipid raft microdomains constitute targets of autoantibodies in POTS patients.

Subcellular fractionation can reduce sample complexity for proteomic analysis and is most efficient when combined with 2-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) studies³. Numerous studies have shown the importance of lipid raft/caveolae microdomains in the regulation of cellular signal transduction, mechanosensing, lipid metabolism, cholesterol homeostasis, and ion channel activities^4–13. Many critical signaling proteins and their effectors are compartmentalized in lipid rafts and if they become autoimmunoreactive, normal cellular functions may be disturbed. These microdomains are richly and tightly packed with cholesterol, sphingolipids, and glycosyl-phosphatidylinositol (GPI) anchored proteins, and thus have a low buoyant density in sucrose gradient fractionations. Caveolae are particularly abundant in terminally differentiated cells such as adipocytes, squamous epithelial cells, and muscle cells¹⁴.

METHODS

Study subjects and IgG isolation

This study was approved by the Mayo Clinic Institutional Review Board and all participants provided written informed consent. Patients were excluded if they had a history of confirmed autoimmune diseases. Seven control subjects (6 females and 1 male, average age 36.1 years) and 10 patients with the diagnosis of POTS (7 females and 3 males, average age 35.1 years) provided 30 ml of venous blood. The detailed clinical and laboratory profiles of the subjects and controls are outlined in Table 1. POTS was diagnosed in the POTS Clinic at the Mayo Clinic under the supervision of Dr. Phillip Low and they satisfied the criteria of such syndrome as previously described¹⁵. IgGs were purified from each serum sample using the Melon gel IgG isolation kit (Pierce Biotechnology). Normal human heart tissue was obtained from the National Disease Research Interchange (NDRI).

Table 1.

Clinical profiles and laboratory findings of controls and POTS patients

	Age	Sex	Allergies	PMH	Medications	Symptoms & Study Findings	ECG HR BPM	Holter HR BPM Avg (range)	Tilt Study Baseline HR (BP)	Tilt Study 10 min HR (BP)	ECHO (LVEF %)
					Controls
1	43	F	Codeine, procaine	Benign thyroid nodule	Levapro
2	23	F
3	45	F		Lipids, LBP, Herpes zoster
4	37	F	Azithromycin, Pseudoephed rine	Lipids
5	44	F	PCN, shellfish	Sinus rhinitis	Advil, fish oil, Imitrex
6	24	F		Asthma	Alavert
7	37	M		HBP, GERD	Lisinopril, Prilosec
					POTS Subjects
1	44	F	Sulfur, Levofloxacin	Melanoma, meningitis, lipids, fibromyalgia, eczema	RetinA cream	ortho intolerance	126	101 (70–169)	99 (122/80)	122 (110/84)	Normal (60–65%)
2	51	F		Bipolar, HBP	Catapres, Metoprolol, Lipitor, Clonidine, Provera	ortho Intolerance, mild auto neuropathy	117	114 (89–165	92 (148/92)	134 (125/76)	Normal (62%)
3	48	M		Fibromyalgia, chronic fatigue, PTSD	Vit D, Lyrica, Imitrex, Inderal, Florinef	ortho intolerance, mild postgang sudomoter impair, mild multifocal anhidrosis	63	73 (50–145)	74 (122/78)	125 (110/70)	Normal (60%)
4	56	M		GI dysmotility	Florinef, Neurontin, K, Midodrine, Mestinon	severe autonomic neuropathy, severe cardiovagal and adrenergic failure, distal anhidrosis, ortho hypotension	67		64 (164/94)	64 (50/−) (1.5 min)
5	35	F		Asthma, lipids, depression	Klonopin, Midodrine, Nadolol, BCP, inhaler	mild vascular adrenergic impairment, minor hypohidrosis	88	93 (64–152)	88 (122/74)	165 (104/82)	Normal (63%)
6	18	F	Hydrocodone	Irritable bowel, hypovitaminosis D	Metoprolol, Citalopram, vit D	ortho tachycardia	88		76 (108/64)	120 (100/80)
7	20	F	Amoxacillin		Allegra, Microgestin	↓ortho tolerance, hyperadrenergic state	92		82 (110/56)	131 (100/70)	Normal (61%)
8	21	F	Tramadol, Sprintec	Mononucleosis, irritable bowel	Florinef, Neurontin, Lexapro, Metoprolol, Prilosec, Mestinon	focal sudomotor failure, ortho intolerance	67	90 (64–137)	72 (92/64)	101 (86/64)
9	26	F	Amoxacillin	Eczema. GERD, sinus, periurethral cyst	Klonopin, Microgestin, Zyrtec, Norco, Cymbalta	ortho intolerance	82	84 (67–137)	83 (114/70)	137 (108/84)
10	32	M		HBP, lipids, arthritis, asthma. GI dysmotility	Flexeril, Florinef, Neurontin, Klonopin, Mestinon, Propranolol	ortho intolerance, mild adrenergic vasoconstr failure	71	87 (54–136)	80 (110/76)	131 (112/78)

Purification of lipid raft fractions from human heart tissue

Lipid raft fractions were prepared by the non-detergent method as we have described¹⁶. Briefly, frozen heart tissue from all four chambers was homogenized and sonicated with 2 ml of 500 mM sodium carbonate (pH11) with protease inhibitors (Roche Diagnostics, Germany). The homogenate was centrifuged at 2,500 × g for 10 min and the supernatant containing 3 mg of proteins was adjusted to 40% sucrose by mixing with 2 ml of 80% sucrose prepared in MBS (0.15 M NaCl, 25 mM 2-[N-Morpholono] ethanesulfonic acid, pH6.5) and placed at the bottom of an ultracentrifuge tube. Discontinuous sucrose gradients of 5% and 30% were layered on top (4 ml of 5% sucrose/4 ml of 30% sucrose, both in MBS containing 250 mM sodium carbonate, pH11). Samples were then centrifuged at 260,000 × g for 20 hours at 4°C. Twelve fractions of 1 ml each were collected sequentially from the top. The lipid raft fractions were identified by immunoblotting against anti-caveolin-3 antibodies (1:1000, BD Transduction labs), dialyzed against ammonium bicarbonate (50 mM, pH7.8) and lyophilized to reduce sample volume.

2DE and immunoblotting

The experiments were performed as we have described². Proteins from the human cardiac lipid raft fractions prepared above were resolved by 2DE and transferred electrophoretically onto a PVDF membrane. IgGs isolated from each control and patient were used to develop a separate immunoblot. Antibodies against Desmin (1:1000, Cell Signaling), HSP70 (1:1000, Enzo Life Sciences) and cavin-1 (1:2000, Bethyl Laboratory, Inc) were used for 2DE gel immunoblotting to identify their spot locations in 2DE gels.

Protein identification and data analysis

Protein spots specifically reacting against patient IgGs were selected and processed for protein identification by in-gel trypsin digestion and nano-LC-electrospray tandem mass spectrometry as previously described².

Protein interaction network analysis

Ingenuity Pathway Analysis (IPA, Ingenuity® Systems) was used to identify signaling pathways and networks as previously described². The composite was laid out graphically using the network visualization algorithm Cytoscape 2.8.2¹⁷.

RESULTS

We first evaluated the effectiveness of lipid raft/caveolae-rich fraction isolation. Caveolin-3, the marker of cardiomyocyte caveolae, was enriched in the low buoyant density fractions (Figure 1). In contrast, the non-lipid raft membrane marker, clathrin, and the subcellular organelle marker Golgi 58, were differentially distributed (Figure 1). Clathrin appeared predominantly in the heavier fractions of the gradient, suggesting the exclusion of clathrin-coated pits as well as clathrin-associated membranes from our caveolae-rich lipid raft fractions. The Golgi apparatus was also distributed in the heavy density fractions. These results suggest that the lipid raft fraction was relatively free of contamination by intracellular organelles.

Figure 1. Immunoblot analysis of human cardiac membrane sucrose density gradient fractions.

(A) An equal volume of each sucrose density gradient fraction was loaded onto each lane. Enrichment of caveolin-3 in the low buoyant density fractions indicated the successful separation of caveolae/lipid raft fraction (top panel). The middle and lower panels show clathrin and Golgi 58, the non-lipid raft plasma membrane and intracellular organelle marker proteins, respectively, are predominantly detected in the heavy fractions, but not in the caveolae-rich fraction. (B) The distribution of protein content across the gradient fractions was expressed as a percentage of the total amount of protein recovered in the gradient.

After 2DE separation and silver staining, 1159 protein spots were visualized (analyzed by PDQuest, Bio-Rad Laboratories, Inc.) (Figure 2A). 2DE gel immunoblotting analysis demonstrated that 34 spots reacted specifically with patient IgGs (Figure 2B& C, Table 2). These spots were further analyzed by nanoLC-MS/MS and the results identified 72 unique proteins (Table 3). A variety of cardiac lipid-raft associated proteins were targeted by the IgGs from POTS patients. These include proteins associated with caveolae structure (cavin), adrenergic signaling (protein kinase A), calcium signaling (sarcalumenin and S100), cytostructures (desmin, desmoplakin, desmoglein, vimentin, and plakoglobin), chaperone (heat shock 70), and energy metabolism.

Figure 2. 2DE gel immunoblotting of human cardiac lipid raft/caveolae fractions.

(A) Protein maps resolved by 2DE with isoelectric focusing, SDS-PAGE and silver staining. (B) Protein spots in representative 2DE immunoblots reactive against purified IgGs from a healthy control (upper panel) and a patient with POTS (lower panel). Arrow shows patient-specific immunoreactive protein spots. (C) Patient-specific immunoreactive spots sampled from silver-stained 2DE gel for protein identification, spots 1 to 34.

Table 2.

POTS patient specific immunoreactive spots

Patient #	Patient specific spots*
009	2, 3, 4
010	2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13
011	5, 6, 7
012	14, 15, 16, 30, 31, 32, 33, 34
013	1
014	17, 18, 19, 30, 31, 2, 3, 4
015	20, 21, 22, 23, 24
016
017
018	3, 4, 30, 31, 25, 26, 27, 28, 29, 12, 13

^*Spot code refer to Figure 2C

Table 3.

Proteins detected from patient specific spots

Symbols	Protein Name	Acc. numbers	Function
S100A8	Protein S100-A8	S10A8_HUMAN	apoptosis
PPP2R1A	Serine/threonine-protein phosphatase 2A	2AAA_HUMAN	apoptosis, chromosome segregation
HSPA9	Stress-70 protein	GRP75_BOVIN	Chaperone, cell proliferation and aging
PDIA6	Protein disulfide-isomerase A6	PDIA6_HUMAN	Chaperone, isomerase
HSPD1	60 kDa heat shock protein	CH60_HUMAN	Chaperone, protein folding
HSPA1A	Heat shock 70 kDa protein 1A/1B	HSP71_HUMAN	Chaperone, protein folding
HSPA8	Heat shock cognate 71 kDa protein	HSP7C_HUMAN	Chaperone, protein folding
LMNB2	Lamin-B2	LMNB2_HUMAN	structural molecule activity, lipodystrophy
TUBA	Tubulin alpha chain	TBA_XENTR	microtubule, structural molecule activity
TUBB2C	Tubulin beta-2C chain	TBB2C_HUMAN	microtubule, structural molecule activity
TUBB5	Tubulin beta-5 chain	TBB5_HUMAN	microtubule, structural molecule activity
ACT	Actin, alpha sarcomeric/cardiac	ACT2_XENLA	cytoskeleton, cell motility
VIM	Vimentin	VIME_HUMAN	cytoskeleton, intermediate filaments
DES	Desmin	DESM_HUMAN	intermediate filament, cardiomyopathy
MYH7	Myosin-7	MYH7_HUMAN	contractile protein, hypertrophic CM
DSP	Desmoplakin	DESP_HUMAN	desmosomal protein, cardiomyopathy
PSMC5	26S protease regulatory subunit 8	PRS8_HUMAN	degradation of ubiquitinated proteins
FHL1	Four and a half LIM domains protein 1	FHL1_HUMAN	muscle development, hypertrophy
FLG2	Filaggrin-2	FILA2_HUMAN	cell differentiation
DSG1	Desmoglein-1	DSG1_HUMAN	mediating cell-cell adhesion
MFGE8	Lactadherin	MFGM_HUMAN	cell adhesion, angiogenesis
JUP	Junction plakoglobin	PLAK_HUMAN	Cell adhesion, cardiomyopathy, arrhythmia
CS	Citrate synthase	CISY_HUMAN	Carbohydrate metabolism, Tricarboxylic acid cycle
ACAA2	3-ketoacyl-CoA thiolase	THIM_HUMAN	apoptosis and mitochondrial damage
CKM	Creatine kinase M-type	KCRM_HUMAN	energy transduction
CKMT2	Creatine kinase S-type	KCRS_HUMAN	energy transduction
ACOT1	Acyl-coenzyme A thioesterase 1	ACOT1_HUMAN	hydrolysis of acyl-CoAs
SLC25A4	ADP/ATP translocase 1	ADT1_HUMAN	exchange of ADP and ATP
ACAT1	Acetyl-CoA acetyltransferase	THIL_HUMAN	ketone body metabolism
HADH	Hydroxyacyl-coenzyme A dehydrogenase	HCDH_HUMAN	Fatty acid metabolism
FH	Fumarate hydratase	FUMH_HUMAN	fumarate metabolic process
MAOB	Amine oxidase	AOFB_HUMAN	metabolism of neuroactive and vasoactive amines
MT-CO2	Cytochrome c oxidase subunit 2	COX2_HUMAN	electron transport
UQCRC1	Cytochrome b-c1 complex subunit 1	QCR1_HUMAN	electron transport, ATP
UQCRC2	Cytochrome b-c1 complex subunit 2	QCR2_HUMAN	electron transport, ATP
SAMM50	Sorting and assembly machinery component 50 homolog	SAM50_HUMAN	mitochondrial function
NDUFS1	NADH-ubiquinone oxidoreductase	NDUS1_HUMAN	NADH oxidation
PDHX	Pyruvate dehydrogenase protein X component	ODPX_HUMAN	pyruvate metabolic, acyltransferase activity
TUFM	Elongation factor Tu	EFTU_HUMAN	Elongation factor
ATP5A1	ATP synthase subunit alpha	ATPA_HUMAN	oxidative phosphorylation
ATP5B	ATP synthase subunit beta	ATPB_HUMAN	oxidative phosphorylation
ATP5C1	ATP synthase subunit gamma	ATPG_HUMAN	oxidative phosphorylation
DECR1	2,4-dienoyl-CoA reductase	DECR_HUMAN	Oxidoreductase
ALDH2	Aldehyde dehydrogenase	ALDH2_HUMAN	Oxidoreductase
DLD	Dihydrolipoyl dehydrogenase	DLDH_HUMAN	Oxidoreductase
DLAT	Dihydrolipoyllysine-residue acetyltransferase- pyruvate dehydrogenase complex	ODP2_HUMAN	Oxidoreductase
DLST	Dihydrolipoyllysine-residue succinyltransferase-2-oxoglutarate dehydrogenase complex	ODO2_HUMAN	Oxidoreductase
ECSIT	Evolutionarily conserved signaling intermediate in Toll pathway	ECSIT_HUMAN	Oxidoreductase
HSDL2	Hydroxysteroid dehydrogenase-like protein 2	HSDL2_HUMAN	Oxidoreductase
IDH2	Isocitrate dehydrogenase	IDHP_HUMAN	Oxidoreductase
LDHA	L-lactate dehydrogenase A chain	LDHA_HUMAN	Oxidoreductase
MDH2	Malate dehydrogenase	MDHM_HUMAN	Oxidoreductase
ACADM	Medium-chain specific acyl-CoA dehydrogenase	ACADM_HUMAN	Oxidoreductase
LDHD	Probable D-lactate dehydrogenase	LDHD_HUMAN	Oxidoreductase
AIFM1	Apoptosis-inducing factor 1	AIFM1_HUMAN	oxidoreductase, apoptosis
VDAC1	Voltage-dependent anion-selective channel protein 1	VDAC1_HUMAN	Oxidoreductase, apoptosis
ACADVL	Very long-chain specific acyl-CoA dehydrogenase	ACADV_HUMAN	Oxidoreductase, cardiomyopathy
PRKAR1A	cAMP-dependent protein kinase type I-alpha regulatory subunit	KAP0_HUMAN	Post-translational modification
PRKAR2A	cAMP-dependent protein kinase type II-alpha regulatory subunit	KAP2_HUMAN	Post-translational modification
LDB3	LIM domain-binding protein 3	LDB3_HUMAN	PKC-mediated signaling
PGK1	Phosphoglycerate kinase 1	PGK1_HUMAN	Kinase, transferase
PKM2	Pyruvate kinase isozymes M1/M2	KPYM_HUMAN	pyruvate, apoptosis, calcium signaling
PTRF	Polymerase I and transcript release factor	PTRF_HUMAN	caveolae formation, cell membrane repair
APOD	Apolipoprotein D	APOD_HUMAN	ligands transport
PACSIN3	Protein kinase C and casein kinase substrate in neurons protein 3	PACN3_HUMAN	vesicle formation and transport
SRL	Sarcalumenin	SRCA_HUMAN	regulation of calcium transport
FGB	Fibrinogen beta chain	FIBB_HUMAN	platelet aggregation
FGG	Fibrinogen gamma chain	FIBG_HUMAN	platelet aggregation
ANXA2	Annexin A2	ANXA2_HUMAN	stress response, exocytosis
ANXA6	Annexin A6	ANXA6_HUMAN	regulate intracellular Ca2+ release
ANXA11	Annexin A11	ANX11_HUMAN	cytokinesis
ANXA7	Annexin A7	ANXA7_HUMAN	membrane fusion, exocytosis

To validate the results from nanoLC-MS/MS, specific antibodies against desmin, cavin-1 and HSP70 were used against 2DE blots of cardiac lipid raft/caveolae fractions (Figure 3). The results are in agreement with those from proteomic analysis, confirming that the protein identification results were correct.

Figure 3. 2DE gel immunoblot of human heart lipid raft/caveolae fractions against anti-cavin-1, -desmin and -HSP70 antibodies.

Immunoblot analyses of 2DE of lipid raft/caveolae fractions against commercially available antibodies (left panel) and against patient serum IgGs (right panel). The results show agreement for anti-cavin-1 (A), anti-desmin (B), and anti-HSP70 (C).

To help understand the physiological and pathophysiological relevance of the immunoreactive lipid raft proteins, we performed network and canonical pathway analysis on the data from Table 3 using the IPA. Seventy-two identified proteins were integrated into a composite neighborhood comprised of 113 nodes linked by 389 interactions or edges (Figure 4). Network topology was assessed as an undirected network and the results demonstrated that the network topology was not random. A nonstochastic property was confirmed by examination of the interrelationship between node degree (k) and degree distribution (P[k]) (Figure 4). The organized assemblage followed a power law distribution falling within the predicted confidence range of biological networks¹⁸.

Figure 4. Network analysis of the proteins identified by MS from patient-specific protein spots.

(A) Seventy-two unique proteins identified by MS submitted to Ingenuity Pathways Analysis as focus nodes were integrated into a composite neighborhood comprised of 113 protein interaction network linked by 389 interactions or edges. Nodes are listed by Swiss-Prot gene designations and the color corresponds to ontological function. (B) Network degree distribution, (P[k]) versus degree (k), followed a power law distribution indicating nonstochastic scale-free network architecture a typical biological networks.

Canonical pathway analysis demonstrated that many of the proteins targeted by patient IgGs are interrelated in pathways based on the IPA library (Table 4). Many pathways are involved including those that regulate caveolae-mediated signaling, oxidative phosphorylation, fatty acid metabolism, protein ubiquitination, and cardiac β-adrenergic signaling (Table 4).

Table 4.

Canonical pathways involved in proteins targeted by autoantibodies

Ingenuity Canonical Pathways	Molecules (Symbols)
Mitochondrial Dysfunction	ATP5C1, NDUFS1, ATP5B, UQCRC2, MT-CO2, UQCRC1, AIFM1
Oxidative Phosphorylation	ATP5C1, NDUFS1, ATP5B, UQCRC2, MT-CO2, UQCRC1, ATP5F1
Pyruvate Metabolism	PKM2, LDHD, DLAT, PDHX, ACAT1
Valine, Leucine and Isoleucine Degradation	ACADVL, ACAT1, ACAA2, HADH
Citrate Cycle	CS, DLST, IDH2
Purine Metabolism	PKM2, ATP5C1, ATP5B, MYH7, HSPD1, ATP5F1
Fatty Acid Metabolism	ACADVL, ACAT1, ACAA2, HADH
Lysine Degradation	DLST, ACAT1, HADH
Extrinsic Prothrombin Activation Pathway	FGB, FGG
Fatty Acid Elongation in Mitochondria	ACAA2, HADH
Glycolysis/Gluconeogenesis	PKM2, DLAT, PDHX
Intrinsic Prothrombin Activation Pathway	FGB, FGG
Alanine and Aspartate Metabolism	DLAT, PDHX
Acute Phase Response Signaling	ECSIT, FGB, FGG
Butanoate Metabolism	ACAT1, HADH
Propanoate Metabolism	ACADVL, ACAT1
IL-1 Signaling	ECSIT, PRKAR2A
Synthesis and Degradation of Ketone Bodies	ACAT1
Cardiomyocyte Differentiation via BMP Receptors	MYH7
AMPK Signaling	CKM, PRKAR2A
Tryptophan Metabolism	ACAT1, HADH
Tight Junction Signaling	PRKAR2A, MYH7
Calcium Signaling	PRKAR2A, MYH7
ILK Signaling	MYH7, DSP
Huntington’s Disease Signaling	ATP5B, HSPA9
Protein Ubiquitination Pathway	HSPA9, HSPD1
Glucocorticoid Receptor Signaling	HSPA9, FGG
Caveolar-mediated Signaling	PTRF
Nitric Oxide Signaling in the Cardiovascular System	PRKAR2A
Arginine and Proline Metabolism	CKM
Melanocyte Development and Pigmentation Signaling	PRKAR2A
α-Adrenergic Signaling	PRKAR2A
Cardiac β-adrenergic Signaling	PRKAR2A
Insulin Receptor Signaling	PRKAR2A
Role of NFAT in Cardiac Hypertrophy	PRKAR2A
cAMP-mediated signaling	PRKAR2A
Cardiac Hypertrophy Signaling	PRKAR2A

DISCUSSION

In this pilot study, we have made the following observations. First, autoantibodies against cardiac lipid raft microdomain proteins are present in patients with POTS. Second, multiple lipid raft/caveolae proteins are potential targets of the autoantibodies in POTS. Third, the presence of autoantibodies may affect the regulation of multiple cellular processes in patients with POTS.

Proteomic technologies have provided ideal tools in the search of antigens and antibodies in autoimmune diseases. The approach combining 2DE gel immunoblotting and MS has been effective in the identification of new antigens/antibodies in autoimmune and other diseases¹⁹. While the analytical power of MS-based technologies has become greatly improved in recent years, sample preparation remains a limitation for optimal sensitivity of proteomic analysis. In extremely complex protein preparations, the presence of high abundance proteins reduces the ability to detect low abundance proteins. To reduce sample complexity and augment low abundance proteins, we prepared lipid raft/caveolae fractions which are abundant in cardiomyocytes. Caveolae are specialized lipid rafts enriched with cholesterol, caveolins, and signaling molecules and they provide platforms for integrating specific cellular signal transduction processes¹³. Interaction between autoantibodies and lipid raft/caveolae proteins may trigger alterations in signaling pathways to produce the cardiovascular abnormalities seen in patients with POTS.

Our results showed that proteins regulating multiple cellular processes, such as signaling, metabolism, oxidoreductases, chaperones, catabolic, cytostructure and transcription, could be the targets of autoantibodies in patients with POTS. Many of the proteins have previously been implicated in cardiac dysfunction or cardiac disease, such as S100A8²⁰, HSP70²¹, desmin²², and desmoplakin²³. However, involvement of these proteins in the pathogenesis of POTS is unknown. Further studies are needed to substantiate the role of these proteins in POTS. Although it is difficult to predict the prevalence of autoantibodies in the POTS population from the limited number of patients studied, our results indicate that a multitude of cardiac lipid raft-associated proteins could be targeted by autoimmunoreactive IgGs in these patients (Table 2). As shown in Table 2, the putative autoantigen profile of each individual patient was variable. Analyses of large population-based data sets are needed to provide further information on these issues.

Validation of the nanoLC-MS/MS results using affinity purified antibodies of desmin, cavin-1 and HSP70 was performed (Figure 3). The results are consistent with those from MS analyses indicating that this technology is a valuable approach in identifying potential autoimmune targets. Desmin, HSP70 and cavin-1 have been shown to be associated with caveolae^24–26 and cardiovascular diseases²². Desmin is a critical component of cardiac myocyte architecture, structure and signaling. Mice without desmin have impaired mitochondrial function with abnormalities in cardiac and skeletal muscles²⁷. These animals are weak and easily fatigable, and the lack of desmin renders myofibers more susceptible to damage during contraction²⁸. Increased plasma levels of anti-HSP70 were found to be a cardiac risk factor associated with a significant incidence of ECG abnormalities characteristic of chronic myocardial ischemia, conduction abnormality, or heart displacement²⁹. Previous study also suggested that higher serum HSP70 antibody level may be a marker for subsequent development of postoperative atrial fibrillation³⁰. Our findings suggest that the presence of autoantibodies against HSP70 could be a risk factor for the development of cardiac dysfunction in POTS.

Interestingly, a newly described protein, cavin-1, also known as polymerase I and transcript release factor (PTRF), is one of the targets of the autoantibodies in POTS. Cavin-1 is required for the formation of caveolae in all mammalian and plays a critical role in regulating caveolae function in endothelial cells²⁶. Cavin-1 also facilitates repair of damaged muscle cells³¹. Cavin-1 mutations in human caused a secondary deficiency of caveolins resulting in muscular dystrophy, as well as arrhythmias and atrial fibrillation³². Therefore, further studies are warranted to investigate the effect of immune reactions against cavin-1.

Cellular proteins are connected through extensive networks of interacting pathways. Assembly of biologically relevant interactomes may help better understand the effects produced by the binding of autoimmunoreactive IgGs to lipid raft/caveolae proteins. The unbiased gene ontology-based network interrogation further indicates that autoimmune reactions to cardiac proteins are biologically relevant with nonrandom effects on cardiac functions (Figure 4). Some of the targeted proteins are important signaling proteins and they encompass many signaling pathways (Table 4). The potential involvement of the β-adrenergic signaling pathway in POTS is particularly intriguing as a putative mechanism that underlies the clinical manifestation in POTS is an imbalance between sympathetic/parasympathetic regulations. Hence, further studies on the functional relevance of autoantibodies and their potential cardiac membrane protein targets may provide important insights into the pathogenesis of POTS. We hope that this study may serve as an impetus for further investigations in this area.

Abbreviations

POTS

postural orthostatic tachycardia syndrome

IPA

Ingenuity Pathway Analysis

2DE

2-dimensional gel electrophoresis

MS

mass spectrometry