HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-Gp120 aptamer

Abstract

HIV-associated cardiomyopathy (HIVCM) is of clinical concern in developing countries because of a high HIV-1 prevalence, especially subtype C, and limited access to highly active antiretroviral therapy (HAART). For these reasons, we investigated the direct and indirect effects of HIV-1 subtype C infection of cultured human cardiomyocytes and the mechanisms leading to cardiomyocytes damage; as well as a way to mitigate the damage. We evaluated a novel approach to mitigate HIVCM using a previously reported gp120 binding and HIV-1 neutralizing aptamer called UCLA1. We established a cell-based model of HIVCM by infecting human cardiomyocytes with cell-free HIV-1 or co-culturing human cardiomyocytes with HIV-infected monocyte derived macrophages (MDM). We discovered that HIV-1 subtype C unproductively (i.e. its life cycle is arrested after reverse transcription) infects cardiomyocytes. Furthermore, we found that HIV-1 initiates apoptosis of cardiomyocytes through caspase-9 activation, preferentially via the intrinsic or mitochondrial initiated pathway. CXCR4 receptor-using viruses were stronger inducers of apoptosis than CCR5 utilizing variants. Importantly, we discovered that HIV-1 induced apoptosis of cardiomyocytes was mitigated by UCLA1. However, UCLA1 had no protective effective on cardiomyocytes when apoptosis was triggered by HIV-infected MDM. When HIV-1 was treated with UCLA1 prior to infection of MDM, it failed to induce apoptosis of cardiomyocytes. These data suggest that HIV-1 causes a mitochondrial initiated apoptotic cascade, which signal through caspase-9, whereas HIV-1 infected MDM causes apoptosis predominantly via the death-receptor pathway, mediated by caspase-8. Furthermore the data suggest that UCLA1 protects cardiomyocytes from caspase-mediated apoptosis, directly by binding to HIV-1 and indirectly by preventing infection of MDM.

Figure 1. Phenotyping cultured human cardiomyocytes.

Human cardiomyocytes cultured on microscope cover slips where phenotyped using cardiac markers (A) α-MHC (63× magnification) (AF546-labeled secondary antibody [red]) (B) Cx43 (x40) (AF488-labeled primary antibody [green]) and (C) cTnT (40× magnification) (AF546-labeled secondary antibody [red]). Cardiomyocytes where imaged on a Zeiss 780 immunofluorescent confocal microscope.

Figure 2. Kinetics of HIV-1 infection of CM and PBMC.

(A) Agarose-gel electrophoresis of the PCR-amplified U3/U5 region of a dual tropic (R5X4) HIV-1 subtype C isolate called HIV-1_CM9 infected culture lysates at different times after infection. The 539 bp fragment indicates the presence of proviral DNA. A 288 bp region of the gene coding for α-tubulin is a control ensuring equal loading of the wells. (B) Proviral DNA in CM exposed to serial dilutions of AZT. (Insert) Agarose-gel electrophoresis of cell-free HIV-1_CM9 culture digested with DNaseI for 1 h prior to infection. (C) Detection of replication of HIV-1_CM9 in CM and PBMC using p24 ELISA.

Figure 3. Quantification of the relationship between HIV-1 tropism and induction of CM apoptosis using TUNEL assay.

Statistically significant TUNEL positive results compared to mock-infected control cells were determined by the student t-test and are marked as: *, P<0.05; ·, P<0.005; °, P<0.0005 (n = 3± SEM).

Figure 4. Cytochrome c release in HIV-1_CM9-infected CM.

CM were either (A) mock infected; (B) HIV-1 infected; (C) infected in the presence of casp8i; (D) infected in the presence of casp9i and (E) infected with virus pre-incubated with UCLA1. Data were presented in dot plots relating fluorescent intensity as a measure of cytochrome c release against forward scatter.

Figure 5. Caspase 8 and 9 activities in CM infected with HIV-1.

CM were exposed to: media alone; conditioned media containing virus; HIV; 100 µM of casp8i; 100 µM of casp9i; conditioned media and 100 µM of casp8i; conditioned media and 100 µM of casp9i; conditioned media and 100 nM UCLA1 aptamer; HIV and 100 µM of casp8i; HIV and 100 µM of casp9i; HIV pre-incubated for 1 h with 100 nM of UCLA1 aptamer. Cells were harvested daily for 7 days. (A) ATP levels were measured as a correlate of cell viability; (B) caspase 8 and (C) caspase 9 activities were measured as indicators of extrinsic and intrinsic apoptosis, respectively. All assays were luminescence-based and presented as graphs relating RLU to number of days (n = 3± SEM).

Figure 6. CM apoptosis in a co-culture model with HIV-infected MDM.

MDM were infected with the R5-tropic viruses SW2, SW4 or the dual tropic viruses CM9, DU179-99 and RP1 for 24 h. Uninfected CM were added and the co-cultures were then incubated for a further 24 h. Following incubation, the cell monolayers were stained for TUNEL detection. Statistically significant TUNEL positive results compared to mock-infected control cells were determined by the student t-test and are marked as: *, P<0.05; ·, P<0.005; °, P<0.0005 (n = 3± SEM).