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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2017 Sep;187(9):1960–1970. doi: 10.1016/j.ajpath.2017.05.011

HIV-Associated Cardiovascular Disease

Role of Connexin 43

Lisa Prevedel ∗,†, Camilla Morocho ∗,†, Michael VL Bennett ‡, Eliseo A Eugenin ∗,†,∗
PMCID: PMC5809504  PMID: 28688235

Abstract

Chronic HIV infection due to effective antiretroviral treatment has resulted in a broad range of clinical complications, including accelerated heart disease. Individuals with HIV infection have a 1.5 to 2 times higher incidence of cardiovascular diseases than their uninfected counterparts; however, the underlying mechanisms are poorly understood. To explore the link between HIV infection and cardiovascular diseases, we used postmortem human heart tissues obtained from HIV-infected and control uninfected individuals to examine connexin 43 (Cx43) expression and distribution and HIV-associated inflammation. Here, we demonstrate that Cx43 is dysregulated in the hearts of HIV-infected individuals. In all HIV heart samples analyzed, there were areas where Cx43 was overexpressed and found along the lateral membrane of the cardiomyocyte and in the intercalated disks. Areas of HIV tissue with anomalous Cx43 expression and localization also showed calcium overload, sarcofilamental atrophy, and accumulation of collagen. All these changes were independent of viral replication, CD4 counts, inflammation, and type of antiretroviral treatment. Overall, we propose that HIV infection increases Cx43 expression in heart, resulting in tissue damage that likely contributes to the high rates of cardiovascular disease in HIV-infected individuals.

HIV/AIDS represents a growing concern with an estimated 36 million cases worldwide and 1.2 million in the United States. The introduction of effective antiretroviral treatment (ART) has led to increased life span of HIV-infected (HIV+) individuals almost becoming a chronic disease. However, the prevalence of cardiovascular disease or cardiomyopathy in the HIV-infected population is 1.5 to 2 times higher than in the uninfected (HIV−) population1, 2, 3, 4 and may be due to chronic inflammation, hypertension, endothelial dysfunction, insulin resistance, toxicity of antiretroviral drugs, and dyslipidemia.5, 6, 7 HIV cardiomyopathy is characterized by loss of cardiomyocytes and their replacement by fibrous tissue and infiltration of immune cells.8, 9, 10 However, the pathogenesis of heart disease in HIV+ individuals remains largely unknown. We propose that the dysregulation of connexin 43 (Cx43) channels play a key role in the pathogenesis of HIV heart disease.

Gap junctions (GJs) allow the exchange of ions, metabolites, second messengers, and small RNAs and serve as low resistance bridges between coupled cells that enable action potential propagation in excitable tissues and coordination of metabolism.11 In the healthy adult heart, Cx43 is abundantly expressed in the myocardium and is mainly located in GJs at the intercalated disks. Alterations in Cx43 expression and/or subcellular distribution in the heart have been associated with several diseases, including hypertension, infarction, ischemia, hypertrophic cardiomyopathy, atrial fibrillation, and arrhythmias.12, 13, 14, 15, 16, 17 However, the effect of HIV on Cx43 expression and distribution in the heart has not been examined. Recently, we described that HIV-tat protein, the trans-activator of the virus, binds to the Cx43 promoter in human astrocytes and enhances Cx43 expression, thereby increasing gap junctional communication and likely the number of open hemichannels.18 It is unknown whether the same mechanism of Cx43 up-regulation occurs in cardiomyocytes. Here, we report that Cx43 is overexpressed and mis-localized in all postmortem heart tissue samples analyzed from HIV-infected individuals. These changes in Cx43 were independent of CD4 counts, HIV replication status, and ART. Furthermore, areas with dysregulated Cx43 expression and distribution also have signs of calcium overload, mitochondrial proliferation, muscle compromise, and fibrosis. Thus, we propose that overexpressed Cx43 is a main contributor to the increased heart disease observed in HIV-infected individuals.

Materials and Methods

Study Participants

Donors were recruited by National NeuroAIDS Tissue Consortium (NNTC) as part of a National Institute of Mental Health and National Institute of Neurological Disorders and Stroke program to collect, store, and distribute samples obtained from HIV+ and HIV− individuals. Uninfected and HIV-infected individuals were uniformly distributed among age and comorbidities (Table 1 and data not shown). The only uneven characteristic of our population was sex, most of our uninfected and HIV-infected samples were obtained from men. In addition, the population analyzed had a broad spectrum of CD4 counts, plasma viral load, ART, and length of HIV infection. The uninfected population analyzed was cognitively normal. In contrast, 49% of the HIV-infected population was neurocognitive normal (28 cases), 18% cognitive impaired (10 cases), 28% was cognitive impaired to other or uncertain cause (16 cases), and 5% was unable to reliably assign neurocognitive diagnostic (3 cases). These data indicated that even HIV-infected individuals had normal cognition, yet they may have significant alterations in heart disease as demonstrated below.

Table 1.

Patient Information

Patient No. HIV status Age, years Sex ART current ARVs (2 years of death) CD4 counts, cells/mm Viral load, copies/mL Time with HIV, years
1 + 45 M 3TC, ABC, APV, CBV, D4T, DDC, DDI, DLV, EFV, HU, IDV, NFV, NVP, RTV, SQV, ZDV 25 124,380 13
2 + 58 M 3TC, ABC, APV, DDI, IDV, NVP, ZDV 87 3806 9
3 + 24 M NR 141 NR <1
4 + 61 M 3TC, RTV, SQV, TFV 69 40 28
5 + 23 M 3TC, DDI, NVP 4 200 1
6 + 55 M 3TC 3 240,337 7
7 + 58 M 3TC, ABC, EFV, KTA, RTV 72 279 <1
8 + 43 M ABC, KTA, NVP, RTV, TFV 12 90,258 16
9 + 47 M 3TC, KTA, NFV, NVP, RTV, TFV, ZDV 199 NR 17
10 + 52 M NR NR NR 16
11 + 51 M FTC, TFV 34 605,555 19
12 + 56 M ATV, EFV, RTV, TFV 365 25,669 16
13 + 50 M ND 253 400 NR
14 + 44 M Yes, NR NR NR 23
15 + 52 M NR 630 40 23
16 + 49 M 3TC, ABC, KTA, RTV, TFV, TZV, ZDV 273 400 10
17 + 44 M NR 78 3484 4
18 + 64 M DRV, EPZ, RTV 299 40 18
19 + 39 M D4T, DDI, NFV NR 400 7
20 + 35 M ABC, APV, D4T 40 250,000 13
21 − 69 M NA NA NA NA
22 − 38 M NA NA NA NA
23 − 51 F NA NA NA NA
24 − 54 M NA NA NA NA
25 − 66 M NA NA NA NA
26 − 74 F NA NA NA NA
27 + 42 M 3TC, D4T, FTV, SQV 101 400 5
28 + 30 M APV, CBV, NVP 115 63,909 9
29 + 49 M NR 14 651,401 <1
30 + 60 M NR 24 61,223 10
31 + 47 M DRV, RTV, TRU 61 48 20
32 + 60 M ATR 398 ND 11
33 + 46 M 3TC, ATR, CBV, EFV, KTA, NFV, RTV 22 ND 7
34 + 59 M 3TC, CBV, D4T, NVP, TRU 158 40 13
35 + 60 M EPZ, KTA 497 48 6
36 + 54 M 3TC, ABC, ATR, ATV, CBV, DRV, EPZ, KTA, NFV, RTV, TRU 384 40 12
37 + 71 M 3TC, ATV, KTA, RTV, TRU, ZDV 403 40 16
38 + 61 M NR 315 NR 20
39 + 39 M DRV, KTA, RTV, TRU 112 40 15
40 + 31 M NR NR 16,032 2
41 + 34 M NR NR 54,148 6
42 + 64 M 3TC, D4T 96 2064 12
43 + 50 M TZV 256 400 15
44 + 40 M NR NR NR 5
45 + 39 M 3TC, ABC, D4T, EFV, IDV, KTA, NVP 13 75,000 14
46 + 59 M 3TC, DDC, DLV, EFV, TFV, ZDV 32 400 12
47 + 37 M NR NR 30,769 9
48 + 43 M NR NR NR NR
49 + 29 F NR NR NR 10
50 + 37 M NR 24 NR 5
51 + 35 M ATV, FTC, RTV, TFV NR NR 11
52 + 52 M 3TC, D4T, NVP 41 400 7
53 + 34 M ATV, RTV, TRU 24 322 6
54 + 58 M NR 5 505,903 23
55 + 52 M RTV, SQV, TRU 317 500 10
56 + 57 M KTA, TRU 518 50 17
57 − 83 M NA NA NA NA
58 + 25 M NR NR NR NR
59 + 46 M NR NR NR NR
60 + 51 M NR NR NR 15
61 − 53 F NA NA NA NA
62 + 70 M EPZ, TMC NR <50 NR
63 − 58 M NA NA NA NA
64 − 43 M NA NA NA NA
65 + 59 M NR 32 83,804 <1
66 + 54 F EFV, TRU 156 50 NR
67 − 63 M NA NA NA NA
68 + 46 M NR 61 50 17
69 − 42 F NA NA NA NA
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F, female; M, male; 3TC, lamivudine (Epivir); ABC, abacavir (Ziagen); APV, amprenavir (Agenerase); ART, antiretroviral treatment; ARV, antiretroviral; ATR, efavirenz 600 mg + emtricitabine 200 mg + tenofivir DF 300 mg (Atripla); ATV, atazanavir (Reyataz); CBV, zidovudine + lamivudine (Combivir); D4T, stavudine (Zerit); DDC, zalcitabine (Hivid); DDI, didanosine (Videx); DLV, delaviridine (Rescriptor); DRV, TMC-114, darunavir (Prezista); EFV, efavirenz (Sustiva); EPZ, ziagen + epivir (Epzicom); FTC, emtricitabine (Coviracil; Emtriva); FTV, SQV2, saquinavir-sgc (Fortovase); HU, hydroxyurea; IDV, indinavir (Crixivan); KTA, LPV/RTV, lopinavir/ritonavir (Kaletra); NA, not applicable; ND, not determined; NFV, nelfinavir (Viracept); NR, not recorded; NVP, nevirapine (Viramune); RTV, ritonavir (Norvir); SQV, saquinavir (Invirase); TFV, tenofivir DF (Viread); TMC, TMC-125, etravirine (Intelence); TRU, emtricitabine + tenofovir (Truvada); TZV, AZT + 3 TC + abacavir; zidovudine + lamivudine + abacavir (Trizivir); ZDV, zidovudine (alias AZT; Retrovir).

Immunofluorescence and Confocal Microscopy

Tissue sections were incubated in blocking solution for 2 hours at room temperature and then in diluted primary antibody (anti-pan Cx43: dilution 1:2000, Sigma C6219; Sigma-Aldrich, St. Louis, MO; or anti–N-cadherin: dilution 1:250, BD 61092; Becton Dickinson, Franklin Lakes, NJ) overnight at 4°C. Tissues were washed several times with phosphate-buffered saline at room temperature and incubated with the appropriate secondary antibody for at least 2 hours at room temperature, followed by another wash in phosphate-buffered saline. Tissues were examined using an A1 Nikon confocal microscope (Nikon, Tokyo, Japan). Antibody specificity was confirmed by replacing the primary antibody with a nonspecific myeloma protein of the same isotype or nonimmune serum as previously described.19, 20 The expression level and distribution of the target protein were analyzed using the three-dimensional reconstructions and deconvolution, followed by the generation of regions of interest. With the use of the corresponding regions of interest the numbers of positive pixels for the whole deconvoluted three-dimensional construction were calculated. The expression measurement was determined by quantifying the intensity of positive pixels for Cx43 and lipofuscin. To compare control and HIV conditions, a similar number of cells (300 to 800 cells) and total area were analyzed. This analysis avoids usual problems of this kind of experiment, such as changes in cell number or excessive inflammation. Details of the procedure, including deparaffinization, antigen retrieval, blocking, immunohistochemical staining with primary and secondary antibodies, and specificity testing, were previously described.19, 21

RNA Isolation and Quantitative RT-PCR

Total RNA was isolated from 10-mm3 pieces of ventricular heart using RNeasy plus universal tissue mini kit from Qiagen (Hilden, Germany) according to the manufacturer's instructions and quantified using a Nanodrop (Thermo Scientific, Wilmington, DE). Synthesis of cDNA was performed using iScript cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Relative mRNA expression of Cx43 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was determined using Sybr green kits (Life Technologies, Carlsbad, CA). Results are represented as a relative expression of Cx43 normalized to that of GAPDH as a housekeeping gene. The ΔCt value was determined by subtracting the average Ct of the housekeeping gene from the average Ct of the Cx43 gene (official name GJA1). Expression of Cx43 mRNA was compared between the uninfected and HIV-infected individuals and analyzed by paired two-tailed t-test.

Western Blot Analysis

Small ventricular heart pieces were homogenized in RIPA buffer (Cell Signaling, Beverly, MA) containing protease inhibitors (Cell Signaling, Danvers, MA), and 40 μg of protein was electrophoresed on a 10% polyacrylamide gel (Bio-Rad) and transferred to nitrocellulose membranes. Membranes were probed with rabbit antibodies to connexin43 (Sigma-Aldrich) and GAPDH (Santa Cruz Technologies, Carlsbad, CA). Densitometric analysis was performed using ImageJ software version 1.51j8 (NIH, Bethesda, MD).

Von Kossa Staining

Human heart tissue frozen sections were fixed in cold ethanol for 15 to 20 minutes. After rinsing, the fixed tissues were incubated with 5% silver nitrate solution (ab150687; Abcam, Cambridge, UK) and cross-linked under UV light for 30 minutes and then rinsed twice with distilled water. The tissue sections were incubated with (5%) sodium thiosulfate solution for 5 minutes, rinsed twice with distilled water, incubated with Nuclear Fast Red solution for 5 minutes, and rinsed three times with distilled water to remove excess stain. Serial sections were used to identify areas with compromised Cx43 expression.

Electron Microscopy

Fresh postmortem samples were fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer, postfixed in 1% osmium tetroxide, followed by 2% uranyl acetate, dehydrated through a graded series of ethanol, and embedded in LX112 resin (LADD Research Industries, Burlington, VT). Ultrathin sections were cut on a Reichert Ultracut UCT, stained with uranyl acetate, followed by lead citrate, and viewed on a JEOL 1200EX transmission electron microscope at 80 kv.

Statistical Analysis

Statistical analyses were performed using Origin Lab software version 7.2 (OriginLab Corporation, Northampton, MA). Analysis of variance was used to compare the different groups.

Results

Description of Our Cohort of Uninfected and HIV-Infected Individuals

Our studies were focused on the regulation of cardiac Cx43 in HIV-infected individuals using postmortem human ventricular cardiac tissue obtained from 69 individuals (12 uninfected and 57 HIV-infected) as described in Table 1.

Our uninfected population was eight men and four women with a mean age of 57.8 ± 13.7 years. Our HIV-infected population was 55 men (96.5%) and two women (3.5%) with a mean age of 47.7 ± 11.5 years and mean duration of HIV infection of 11.5 ± 6.7 years, 6.89 ± 1.57 × 104 HIV RNA copies/mL, and 157.3 ± 164.61 CD4 cells/mm3 (Table 1). At the time of death, most of the HIV+ individuals were on ART that included protease inhibitors (Table 1), which have been reported to have intrinsic endothelial toxicity.22, 23 Most of the individuals whose cardiac tissue was analyzed had been followed by the National NeuroAIDS Tissue Consortium (Table 1).

HIV-Infected Individuals Have Increased Expression and Mislocalization of Cx43

Ventricular tissue sections were stained for Cx43 to analyze expression and distribution by confocal microscopy. Normally, Cx43 mainly localizes at contacts between myocytes at the intercalated disks.16, 24, 25, 26, 27 Cx43 containing GJs form a low resistance bridge between cardiomyocytes that mediate action potential propagation and metabolic communication along the muscle fiber. Changes in expression and localization of GJs underlie several forms of heart disease.8, 12, 14, 16, 26, 28, 29, 30, 31, 32, 33, 34, 35 Our data confirmed that Cx43 in the heart from uninfected individuals was mainly localized at the intercalated disks as expected (n = 12) (Figure 1A). However, in all HIV-infected individuals analyzed, irrespective of HIV replication status, CD4 counts, and antiretroviral used, Cx43 staining was increased compared with uninfected individuals (n = 57; P = 0.001; HIV compared with uninfected samples) (Figure 1, A and B). Furthermore, the increased Cx43 in hearts of HIV-infected individuals was distributed predominantly along the lateral margins of the muscle fibers instead of at the intercalated disks (P = 0.05) (Figure 1, A and D). Lateralization of Cx43 is associated with several heart diseases such as ischemia, reperfusion injury, and arrhythmias.8, 12, 14, 16, 26, 28, 29, 30, 31, 32, 33, 34, 35 Interestingly, increased expression of Cx43, its lateralization, and lipofuscin accumulation was present in all HIV-infected individuals analyzed (P = 0.0003) (Figure 1, A and C), suggesting chronic heart damage. Lipofuscin accumulation is also associated with accelerated aging (Figure 1, A and C). Thus, we propose that increased Cx43 expression and lateralization of the protein in HIV-infected individual results in damage to the heart.

Figure 1.

Figure 1

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Expression of connexin 43 (Cx43) in human hearts is up-regulated in HIV-infected individuals compared with heart tissues obtained from uninfected individuals. Postmortem ventricular heart tissue was obtained at the time of autopsy from uninfected and HIV-infected individuals (HIV+) as described in Table 1. Because of the limited numbers of uninfected hearts, two to three distinct areas were quantified. A: Immunohistochemistry was performed using paraffin-embedded and fresh tissues and analyzed by confocal microscopy. Representative tissue staining for nuclei (DAPI), Cx43 (fluorescein isothiocyanate), and lipofuscin (red) in uninfected and HIV-infected individuals. Arrows indicate the Cx43 lateralization in the tissues. Cx43 was up-regulated at the intercalated disks and in the lateral membrane of the cardiomyocytes in all individuals analyzed. B: Quantification of Cx43-positive pixels in human ventricular tissue sections from uninfected and HIV-infected individuals. C: Quantification of the total number of lipofuscin-positive pixels was obtained in uninfected and HIV-infected individuals. D: Quantification of Cx43 containing lateral plaques in cardiac ventricular tissue sections obtained from uninfected and HIV-infected individuals. Data are expressed as means ± SD. n = 69 samples (A); n = 3 tissue sections per individual (B and D); n = 2 tissue sections per individual (C). Scale bar = 200 μm. A.U., arbitrary unit.

To confirm that HIV infection induced the lateralization of Cx43 in cardiomyocytes, we stained for Cx43 and N-cadherin to better define the three-dimensional structure of the intercalated disk as previously reported.36 In uninfected conditions 68.8% ± 8.5% of Cx43 colocalized with N-cadherin at the intercalated disks (Figure 2). In the HIV-infected condition, only 32.65% ± 12.02% of Cx43 colocalized with N-cadherin, supporting the hypothesis that Cx43 was mis-localized into lateral membranes of the cardiomyocytes (P = 0.0007 versus uninfected tissues) (Figure 2).

Figure 2.

Figure 2

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Lateralized connexin 43 (Cx43) does not colocalize with N-cadherin in heart tissues obtained from HIV-infected individuals. Postmortem ventricular heart tissue was used. Immunohistochemistry was performed using paraffin-embedded and fresh tissues and analyzed by confocal microscopy and three-dimensional reconstruction to examine colocalization of Cx43 and N-cadherin. A: Representative tissue staining for nuclei (DAPI), Cx43 (fluorescein isothiocyanate), and N-cadherin (cyanine 5) in uninfected and HIV-infected tissues. Arrows indicate lateralized Cx43 that also lacks staining for N-cadherin. In heart tissues obtained from uninfected individuals, most Cx43 localizes at the intercalated disk. In contrast, in heart tissues obtained from HIV-infected individuals, most Cx43 does not colocalize with N-cadherin and mainly localizes at the lateral membrane of the cardiomyocyte. B: Quantification of Cx43-positive pixels that colocalize with N-cadherin–positive pixels in HIV− (control) and HIV+ conditions. Data are expressed as means ± SD. Scale bar = 38 μm.

To further demonstrate that HIV infection increased the expression of Cx43 in heart tissues, we performed quantitative RT-PCR and Western blot analysis of cardiac tissue obtained from HIV patients as described in Table 1. Both Cx43 mRNA (n = 57; P = 0.03) (Figure 3, A and B) and protein (n = 12; P = 0.05) (Figure 3, C and D) were increased compared with heart tissue obtained from uninfected individuals. Our data confirmed the confocal data that Cx43 mRNA and protein increased in response to HIV infection in all cases analyzed. Further analysis of the HIV population described in Table 1 did not find any correlation between increased Cx43 (mRNA and protein) with type of antiretroviral treatment, CD4 counts, or viral load. The only feature that correlated with increased expression and localization of Cx43 was HIV infection.

Figure 3.

Figure 3

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HIV infection increases expression of connexin 43 (Cx43) mRNA and protein in human ventricular heart tissue. A: Representative quantitative RT-PCR (RT-qPCR) amplifications for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Cx43. B: Quantification of the RT-qPCR experiments for tissues from uninfected and HIV-infected hearts. HIV infection (independent of antiretroviral treatment, CD4 counts, replication, and associated inflammation) increases Cx43 expression (mRNA and protein). Each point represents one individual. C: Representative Western blot analysis of Cx43 and GAPDH protein expression in HIV− and HIV+ hearts. GAPDH was used as a loading control; mouse astrocytes were used as a positive control for Cx43 and its phosphorylation (data not shown). D: Quantification of the Western blots for heart tissues obtained from uninfected and HIV-infected individuals. Each point represents one individual. Data are expressed as means ± SD. CT, cycle threshold.

Calcium Is Dysregulated in Ventricular Areas with Compromised Cx43 Expression and Localization

To examine whether changes in Cx43 localization and expression observed in heart tissue from HIV-infected individuals altered cardiomyocyte function, calcium metabolism was examined using Von Kossa staining.37, 38, 39 To perform these studies, serial tissue sections containing areas with normal and compromised Cx43 expression and localization were selected. Calcium normally diffuses through GJs31, 40, 41, 42 and is sequestered into the mitochondria and endoplasmic reticulum or is extruded from the cell.43, 44, 45, 46 In sections from uninfected individuals, Von Kossa staining was minimal, suggesting a normal calcium metabolism and diffusion (Figure 4A). In contrast, in tissue sections from HIV+ individuals different degrees of pathologic calcium accumulation were observed (Figure 4A). Calcium overload was particularly evident in areas with overexpressed and lateralized Cx43 and pronounced lipofuscin staining (Figure 4A). Quantification of the calcium staining indicated that HIV-infected tissues had an increased calcium staining compared with control uninfected tissues (n = 16; P = 0.005 versus uninfected samples) (Figure 4B).

Figure 4.

Figure 4

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Areas with higher connexin 43 (Cx43) expression and mis-localization show calcium overload. Von Kossa staining to detect calcium deposits in heart sections from uninfected and HIV-infected individuals was performed. All these sections correspond to serial sections in which Cx43 was up-regulated or mis-localized. Sections stained for DAPI, Cx43, and lipofuscin (autofluorescence), and the merge, correspond to the same tissue section. The adjacent tissue section was stained for Von Kossa (labeled calcium). A: Staining for tissue sections obtained from uninfected individuals (uninfected) showing normal Cx43 distribution and low accumulation of lipofuscin with minimal calcium deposits. Staining for tissue sections obtained from HIV-infected individuals (HIV+) showing increased expression of Cx43 in several areas of the tissue and increased accumulation of lipofuscin also shows increased accumulation of calcium deposits in vast areas of the tissue (different degrees of brown-back staining). B: Quantification of calcium staining (dark staining) indicates an increase of 60% in Von Kossa staining. Data are expressed as means ± SD. n = 29 different individuals (A and B). Scale bar = 200 μm. A.U., arbitrary unit.

Ventricular Regions with Altered Cx43 Expression and Localization Show Muscle Compromise and Mitochondrial Proliferation Compared with Areas without Cx43 Dysregulation

Transmission electron microscopy was used to further evaluate the damage in the heart tissue. Two representative examples each of heart tissues obtained from an uninfected and an HIV-infected individual are shown in Figure 5, A and B. In all HIV+ tissues analyzed, there was an increase in the number of mitochondria (P = 0.00003) (Figure 5, C–E), especially in areas with compromised Cx43 expression and localization. In addition, significant decrease in myofibril width in areas with compromised mitochondria (Figure 5, C, D, and F) was detected. Quantification of the myofibril diameter clearly indicated that HIV-infected individuals have two different populations of fibers: myofibrils of normal diameter and myofibrils of about half that diameter compared with uninfected tissues (P = 0.01) (Figure 5F). No significant differences were observed if the two populations of myofibers were quantified compared with the diameter of uninfected myofibers. These compromised myofibrils were associated with the proliferation and altered distribution of mitochondria (P < 0.001 versus uninfected conditions, n = 39).

Figure 5.

Figure 5

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Electron microscopic analysis of hearts obtained from HIV-infected individuals shows that areas with compromised connexin 43 (Cx43) localization and calcium metabolism have extensive mitochondrial proliferation and decreased myofibril width. Areas with normal and increased Cx43 expression were identified and subjected to electron microscopy analysis. A and B: Representative sections from an HIV− heart. Black lines with circles indicate myofibril width. C and D: Representative sections from an HIV+ heart showing mitochondrial proliferation (arrows) and a decrease in myofibril width (black lines with circles). E: Quantification of mitochondrial numbers in heart tissue showing a significant increase in HIV+ tissues. F: Quantification of myofibril width in heart tissues obtained from uninfected and HIV-infected individuals. There are no statistically significant differences between uninfected and HIV-infected individuals in myofibril width. However, analysis of tissues obtained from HIV-infected individuals indicates the presence of two distinct populations of myofibrils, one with normal width and a compromised one that is significantly different that that uninfected individual. Data are expressed as means ± SD. n = 5 grids per patient from five different individuals were analyzed (F). P = 0.01, HIV small myofibers compared with uninfected conditions (F). Scale bar = 1.9 μm.

To further examine the damage in heart tissue obtained from HIV-infected individuals, trichrome stain for detection of collagen fibers and fibrin and periodic acid-Schiff for detection of polysaccharides was performed. Heart tissues obtained from uninfected cells have minimal trichrome staining, especially in blood vessels (Figure 6A). In contrast, HIV+ heart tissue showed increased muscle disorganization as described above and increased accumulation of collagen or fibrin between the muscle fibers (Figure 6A). Quantification of the trichrome staining indicated a significant increase in accumulation of collagen or fibrin between cardiomyocytes in areas with compromised Cx43 expression and distribution (P = 0.001 versus uninfected samples) (Figure 6B). No significant changes in periodic acid-Schiff staining were detected between uninfected and HIV-infected tissue sections (data not shown). In summary, significant damage was observed in areas with compromised Cx43 expression and localization.

Figure 6.

Figure 6

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HIV infection increases the accumulation of collagen/fibrin in areas with increased expression and mis-localized connexin 43 (Cx43). Trichrome staining (blue) was performed to detect accumulation of collagen and fibrin in serial sections with areas already identified to have increased and mis-localized Cx43 expression. A: Trichrome staining of tissue sections obtained from uninfected and HIV-infected individuals. B: Quantification of trichrome staining from individuals indicates that HIV infection increases tissue scaring. n = 10 uninfected individuals (A and B); n = 19 HIV-infected individuals (A and B). Scale bar = 200 μm. A.U., arbitrary unit. Data are expressed as means ± SD.

Discussion

Our data demonstrate that HIV increases Cx43 expression at the intercalated disk and at the lateral membrane of the cardiomyocytes. We found that ventricular areas with Cx43 overexpression and mis-localization also have high calcium levels, increased fibrous tissue, and the proliferation of mitochondria. Thus, we propose that HIV-mediated up-regulation of Cx43 contributes to the onset and progression of heart disease in HIV+ individuals.

Despite the clinical importance of HIV heart disease the cause and contributing factors are poorly understood. Several laboratories using different models have demonstrated that HIV infection in combination with systemic inflammation and ART cytotoxicity results in cardiac pathologic processes.47, 48, 49 Furthermore, in the past couple of years, there is increasing clinical and experimental evidence that HIV infects cardiomyocytes in vitro and in vivo.50, 51, 52 Furthermore, it was reported that gp120 interaction with CXCR4 on the surface of cardiomyocytes triggers caspase-dependent mitochondrial activation and apoptosis.53 In addition to the potential direct effect of HIV in cardiomyocytes, several indirect mechanisms are also responsible for HIV heart damage, including an increase in proinflammatory cytokines, nitric oxide, and inducible nitric oxide synthase, which are released mainly by macrophages.54, 55, 56 In conclusion, several laboratories agree that the damage is multifactorial. Here, we propose that dysregulation of Cx43 also plays a role in HIV-associated cardiotoxicity.

Cx43 is an essential protein in heart development and function.12 In the normal heart, Cx43 is localized to intercalated disks, and little is found in the lateral membranes. Increased lateralization is often associated with reduced conduction velocity and increased in fibrosis.12, 14, 16, 25, 30, 31, 57 Normally, under inflammatory and infectious conditions Cx43 expression and GJ communication is reduced or shut down.11, 58 However, our data on astrocytes in the context of HIV infection indicate that the HIV protein, tat, the transactivator of the virus, binds to the Cx43 promoter, increasing Cx43 expression and gap junctional coupling to enable the spread of toxic intracellular second messengers among coupled cells, resulting in bystander apoptosis and inflammation.18, 58 HIV-tat, like other viral proteins, is secreted into the extracellular space even in the presence of effective ART59, 60 and may enter and affect cells that do not express HIV-tat. Our data indicate that in all of the heart tissues analyzed from HIV-infected individuals Cx43 was increased in abundance and was anomalously distributed along the lateral membranes. The lateral localization of Cx43 protein and the separation between the different muscle fibers suggest that Cx43 hemichannels may be an additional source of cellular compromise and inflammation as described in other systems.58, 61, 62 Opening of Cx43 hemichannels would increase Ca2+ influx into cardiomyocytes and allow Na+ influx, and K+ efflux and loss of ATP and other metabolites into the extracellular space. In agreement with the suggestion of increased hemichannel opening, our data indicate that all HIV-infected individuals have increased circulation of intracellular molecules transported by the opening of hemichannels. Furthermore, HIV infection of astrocytes and cell lines susceptible to HIV infection also results in the opening of Cx43-containing hemichannels.18, 58, 63 Thus, further studies are required to examine the role of Cx43 hemichannels in HIV-associated heart disease.

The increased accumulation of lipofuscin indicates accelerated aging processes in the HIV-infected population as described in other HIV-related pathologic conditions.64 Lipofuscin is a brownish pigment generated by the breakdown and absorption of damaged blood cells or lipids and is associated with aging. Thus, higher amounts of lipofuscin are representative of accelerated aging and/or alterations in lipid metabolism. Studies in mice indicate that aging or senescence normally decreases Cx43 expression and function in the heart,65 a condition different from the increase observed in our study. Furthermore, studies using SIV-infected monkeys indicate a critical role of CCR5 in gp120-mediated inflammation, recruitment of monocytes/macrophages into the cardiac tissue, and compromise of cardiac muscle and function.66, 67, 68, 69 Thus, we propose that an additional mechanism of heart disease in the HIV-infected population is altered in Cx43 expression, distribution, and function. Several groups also demonstrated the presence of Cx43-containing channels on the surface of the mitochondria,12, 70, 71 but the role of this mitochondrial Cx43 in cardiomyopathy is still under investigation. Our data indicate that both astrocytes and macrophages have compromised and enlarged mitochondria, resulting in significant changes in metabolism and bystander killing mechanisms mediated by connexin-containing channels, GJs, and hemichannels. Future studies will address these intriguing possibilities.

Conclusions

We consistently detected increased Cx43 mRNA and protein, altered localization of the protein, calcium accumulation, and deposition of collagen/fibrin in areas with Cx43 overexpression. All of these effects were independent of age, ART (including protease inhibitors), CD4 counts, viral load, and years living with HIV. Our studies in the brain and heart tissue indicate that HIV uses or targets Cx43 to spread toxicity or inflammation. In the heart, it is not known whether Cx43 up-regulation by HIV occurs during early infection or is a result of chronic damage by HIV and ART. Our data suggest that Cx43 channels are a main target of HIV infection in the body, including the brain and the heart. The targeting of Cx43 in all HIV-infected individuals provides a common mechanism of damage and a potential target to reduce or prevent heart disease in the HIV-infected population.

Acknowledgments

We thank the National NeuroAIDS Tissue Consortium (NNTC) for providing all samples, Dr. Susan Morgello for reading the manuscript, and Dr. Karen Maass (New York University) for providing the N-cadherin antibody.

Footnotes

Supported by the NIH grants MH096625 and NS105584 (E.A.E.) and Public Health Research Institute funding (E.A.E.). The National NeuroAIDS Tissue Consortium is made possible through funding from the National Institute of Mental Health and National Institute of Neurological Disorders and Stroke by the Manhattan HIV Brain Bank grant U24MH100931, Texas NeuroAIDS Research Center grant U24MH100930, National Neurological AIDS Bank grant U24MH100929, California NeuroAIDS Tissue Network grant U24MH100928, and Data Coordinating Center grant U24MH100925.

Disclosures: None declared.

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