Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis

Abstract

Background: Hyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes.

Methods: H9c2 cells (rat cardiomyoblast cells) and adult rat cardiomyocytes were incubated with Hcy and were analyzed for cell viability. Furthermore, we determined the effects of Hcy on intracellular mediators related to cell viability in cardiomyocytes, namely NOX2, reactive oxygen species (ROS), mitochondrial membrane potential (DeltaPsi (m)) and ATP concentrations.

Results: We found that incubation of H9c2 cells with 0.1 mM D,L-Hcy (= 60 microM L-Hcy) resulted in an increase of DeltaPsi (m) as well as ATP concentrations. 1.1 mM D,L-Hcy (= 460 microM L-Hcy) induced reversible flip-flop of the plasma membrane phospholipids, but not apoptosis. Incubation with 2.73 mM D,L-Hcy (= 1.18 mM L-Hcy) induced apoptosis and necrosis. This loss of cell viability was accompanied by a thread-to-grain transition of the mitochondrial reticulum, ATP depletion and nuclear NOX2 expression coinciding with ROS production as evident from the presence of nitrotyrosin residues. Notably, only at this concentration we found a significant increase in S-adenosylhomocysteine which is considered the primary culprit in HHC.

Conclusion: We found concentration-dependent effects of Hcy in cardiomyocytes, varying from induction of reversible flip-flop of the plasma membrane phospholipids, to apoptosis and necrosis.

Fig. 1

Effect of Hcy on cell-viability via flow-cytometry: Flow cytometry analysis of H9c2 cells incubated with different concentrations of d,l-Hcy during 24 h, with or without 25 μM Z-VAD FMK, a pan caspase inhibitor. A total of 10,000 cells were measured (n = 3). (A) Percentage of single annexin V positive, PI-negative labeled cells, which is a marker of flip flop of the plasma membrane phospholipids. (B) Percentage of double-annexin-V/PI-positive cells depicting late apoptotic and/or necrotic cells. Data represent mean percentages and standard error of the mean (SEM)

Fig. 2

Effect of Hcy on caspase-3 activity: H9c2 cells were incubated during 24 h with different concentrations of d,l-Hcy in the presence and absence of Z-VAD FMK (n = 9). Caspase-3 activity was measured as indicated under Methods. Data represent mean and SEM

Fig. 3

Effect of Hcy on NOX2 expression: (A) Western Blot analysis for NOX2 expression of whole-cell lysates from H9c2 cells incubated with or without Hcy for 24 h. Data represent mean and SEM for three independent experiments. (B) Western Blot analysis of the same blots for pan-actin as a loading control

Fig. 4

Effect of Hcy on intracellular localization of NOX2 and nitrosylation in H9c2 cells: Analysis of the effect of different concentrations of D,L-Hcy at 24 h incubation on the nuclear localization of NOX2 expression (A) and nitrotyrosine residues (B) measured by means of digital imaging microscopy. The arbitrary units are defined as sum intensity per nucleus and depicted as % increase versus control where control was set to 0 (n = 6). Data represent mean and SEM

Fig. 5

Effect of Hcy on intracellular localization of NOX2 and ROS production in adult rat cardiomyocytes: Digital Imaging microscopy pictures of adult rat cardiomyocytes incubated with 0.1 mM d,l-Hcy during 24 h. Red color (A) indicates NOX2 expression which is visible in the nucleus and in the cytosol (arrow). Green color (B) represents nitrotyrosine formation. The merge image (C) also shows blue DAPI staining for DNA which shows co-localization of NOX2 expression and nitrotyrosine formation in the nucleus. (D) Amount of nuclear NOX2 expression in adult rat cardiomyocyt in control, 0.07 mM and 0.1 mM d,l-Hcy samples (n = 3). (E) Amount of nitrotyrosine formation in adult rat cardiomyocyt in control, 0.07 mM and 0.1 mM d,l-Hcy sample (n = 3). The arbitrary units are defined as sum intensity per nucleus and depicted as % increase versus control where control was set to 0. Data represent mean and SEM

Fig. 6

Effect of Hcy on ATP levels: Increase or decrease in ATP levels (as compared to control levels) in H9c2 cells after incubation with different concentrations of d,l-Hcy during 24 h. Data represent mean and SEM of four independent experiments

Fig. 7

Effect of Hcy on mitochondrial membrane potential: H9c2 cells were incubated with or without Hcy for 24 h and analyzed with digital imaging microscopy. Green indicates the JC-1 monomers. Red indicates JC-1 aggregates, which are formed in the mitochondria when a sufficiently high membrane potential is reached. (A) H9c2 cells incubated in growth medium alone. Normal heterozygous population of elongated and round mitochondria have a red fluorescence signal. (B) H9c2 cells incubated with 0.1 mM d,l-Hcy. An increase in actively respiring mitochondria (red) is visible, and a mostly elongated morphology can be seen. (C) 1.1 mM d,l-Hcy shows a similar amount of functioning mitochondria in the cells that did not receive Hcy; only the morphology of the mitochondria is mostly round, a phenomenon which is known as thread-to-grain transition. (D) H9c2 cells incubated with 2.7 mM d,l-Hcy. Some cells have no functioning mitochondria left, but overall there is no significant loss of red signal. The thread-to-grain transition of the mitochondrial reticulum is complete. (E) Analysis of sum intensity in Cy3 signal (red) per condition as an indicator for ΔΨ_m. Data represent mean and SEM (n = 3)

Fig. 8

Effects of different concentrations of Hcy on cardiomyocyte viability Scheme of the proposed events caused by Hcy, which ultimately lead to cell death: (A) control cells. (B) 0.1 mM d,l-Hcy results in increase of ΔΨ_m and ATP levels, and translocation of NOX2 to the nucleus. (C) 1.1 mM d,l-Hcy results in flip-flop of the plasma membrane (bended arrows in plasma membrane indicate flip-flop of phophatidyl serine to the outer leaflet of the membrane), coinciding with localization of NOX2 in the nucleus. (D) 2.7 mM d,l-Hcy next to flip-flop and NOX2 localization in the nucleus, nuclear ROS production is found. ATP is then decreased, as well as ΔΨ_m. Caspase-3 is activated and PI can enter the cell indicating cell death