Restoration of contractility in hyperhomocysteinemia by cardiac-specific deletion of NMDA-R1

Abstract

Homocysteine (HCY) activated mitochondrial matrix metalloproteinase-9 and led to cardiomyocyte dysfunction, in part, by inducing mitochondrial permeability (MPT). Treatment with MK-801 [N-methyl-d-aspartate (NMDA) receptor antagonist] ameliorated the HCY-induced decrease in myocyte contractility. However, the role of cardiomyocyte NMDA-receptor 1 (R1) activation in hyperhomocysteinemia (HHCY) leading to myocyte dysfunction was not well understood. We tested the hypothesis that the cardiac-specific deletion of NMDA-R1 mitigated the HCY-induced decrease in myocyte contraction, in part, by decreasing nitric oxide (NO). Cardiomyocyte-specific knockout of NMDA-R1 was generated using cre/lox technology. NMDA-R1 expression was detected by Western blot and confocal microscopy. MPT was determined using a spectrophotometer. Myocyte contractility and calcium transients were studied using the IonOptix video-edge detection system and fura 2-AM loading. We observed that HHCY induced NO production by agonizing NMDA-R1. HHCY induced the MPT by agonizing NMDA-R1. HHCY caused a decrease in myocyte contractile performance, maximal rate of contraction and relaxation, and prolonged the time to 90% peak shortening and 90% relaxation by agonizing NMDA-R1. HHCY decreased contraction amplitude with the increase in calcium concentration. The recovery of calcium transient was prolonged in HHCY mouse myocyte by agonizing NMDA-R1. It was suggested that HHCY increased mitochondrial NO levels and induced MPT, leading to the decline in myocyte mechanical function by agonizing NMDA-R1.

Fig. 1.

Phenotyping of cardiac-specific knockout (KO) of N-methyl-d-aspartate (NMDA)-receptor 1 (R1). A: ventricular myocytes were isolated, permeabilized, and processed for confocal microscopy for NMDA-R1 expression. A representative confocal image of myocyte NMDA-R1 expression is presented. B: total ventricular myocyte protein was isolated and processed for immunoblot analysis for NMDA-R1 expression. Data are representative of at least two different experiments (n = 4 mice/group for each experiment).

Fig. 2.

Homocysteine (HCY) induces the production of reactive oxygen species (ROS) and nitric oxide (NO) in the myocyte mitochondria by agonizing NMDA-R1. A: myocytes were isolated and processed for immunoconfocal staining with the mitochondrial-specific, redox-sensitive fluorophore dihydrorhodamine 123 (DHR). The images were acquired and quantitated using ImagePro image analysis software. Values are mean fluorescence intensity ± SE; n = 57–58 myocytes from 4–5 mice/group (n = 17–18 cells/group). P < 0.05 vs. wild-type (WT) mice (*) and vs. NR1^fl/fl/Cre with HCY (#). To determine the mitochondrial purity, the mitochondrial and cytosolic fractions were probed with prohibitin (mitochondrial-specific protein; glyceraldehyde-3-phosphate dehydrogenase, cytosolic marker). A representative Western blot is presented. B: myocyte mitochondria was isolated, 100 μM of l-arginine [mitochondrial nitric oxide synthase (NOS) substrate] were added at the indicated times, and NO levels were recorded using NO-sensitive probe. Calcium dependency of mitochondrial (mt) NOS was determined by the addition of 10 mmol/l EDTA at the end of experiment. NO levels were plotted as pV against time (s). P < 0.05 compared with WT (*) and compared with NR1^fl/fl/Cre with HCY (#). Data are representative of at least two different experiments (n = 6/group).

Fig. 3.

Cardiomyocyte-specific KO of NMDA-R1 attenuate HCY-induced mitochondrial permeability transition and involves matrix metalloproteinase (MMP) activity. Myocyte mitochondria were isolated and suspended in MOPS swelling buffer (pH 7.4). CaCl₂ was added to initiate the swelling, and the absorbance was recorded at 540 nm (A₅₄₀). ΔA₅₄₀ was calculated and plotted. (ΔA₅₄₀ = A_540max − A_540min). Mice were injected ip with cyclosporin (CsA) and, 1 h later, myocyte mitochondria were isolated and processed for swelling assay. P < 0.05 compared with WT (*) and compared with NR1^fl/fl/Cre with HCY and treatment (#). Data represent two different experiments (n = 6/group).

Fig. 4.

Cardiomyocyte-specific deletion of NMDA-R1 restores the HCY-induced decrease in myocyte contractile performance: ventricular cardiomyocytes were field stimulated at 1 Hz, and their mechanical properties were measured. A: data representative of cell shortening. B: graphical presentation of %cell shortening. C: graphical presentation of maximal rate of relaxation and contraction (−dL/dt, +dL/dt). D and E: graphical presentation of time to 90% peak shortening (TPS-90) and relaxation (TR), respectively. Values are means ± SE; n = 57–58 myocytes from 4–5 mice/group (n = 17–18 cells/group). P < 0.05 vs. WT mice (*) and vs. NR1^fl/fl/Cre with HCY and treatments (#).

Fig. 5.

HCY decreases contraction amplitude at high calcium concentration by agonizing NMDA-R1. Ventricular myocytes were isolated, and the contraction amplitudes were compared at 3 different increasing concentrations of CaCl₂ (1–4 mmol/l). The contraction amplitude was plotted as a line graph. Data were analyzed off-line with IonWizard software from IonOptix. Values are means ± SE; n = 57–58 myocytes from 4–5 mice/group (n = 17–18 cells/group). *P < 0.05 vs. WT mice.

Fig. 6.

Cardiomyocyte-specific KO of NMDA-R1 ameliorated the HCY-induced alteration in calcium transient. Adult ventricular myocytes were field stimulated at 1 Hz, and the calcium transients were measured in fura 2-AM-loaded myocytes using IonOptix. The recovery of calcium transient (in terms of tau) was plotted as a bar graph. Time to reach 90 and 50% baseline fluorescence was recorded. Data are means ± SE. *P < 0.05 vs. WT.

Fig. 7.

Schematic presentation of hypothesis. The elevated levels of HCY increase mitochondrial ROS and NO production. That may activate MMP in the mitochondria (16) and induces mitochondrial permeability transition, leading to the decline in myocyte mechanical function by agonizing NMDA-R1.