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American Journal of Physiology - Heart and Circulatory Physiology logoLink to American Journal of Physiology - Heart and Circulatory Physiology
. 2009 Jan 30;296(3):H887–H892. doi: 10.1152/ajpheart.00750.2008

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

Karni S Moshal 1, Munish Kumar 1, Neetu Tyagi 1, Paras K Mishra 1, Naira Metreveli 1, Walter E Rodriguez 1, Suresh C Tyagi 1
PMCID: PMC2660230  PMID: 19181966

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.

Keywords: mitochondrial matrix metalloproteinase, myocyte mechanics, calcium transient, mitochondrial permeability, N-methyl-d-aspartate receptor 1

homocysteine (HCY) is a nonprotein sulfur-containing amino acid formed during the metabolism of dietary methionine. The elevated plasma level of HCY is an independent risk factor for heart failure (4, 6) and for sudden cardiac death (SCD) (1–3).

Although N-methyl-d-aspartate receptor-1 (NMDA-R1) expression has been widely studied in the central nervous system, evidence for its expression in nonneuronal tissue is rare. NMDA-R1 is expressed in cardiomyocytes (8, 9) and endothelial cells (18). The NMDA-R1 antagonist dizocilpine ameliorates stress-induced sudden death in cardiomyopathic hamster (12). The cardiomyocyte expresses NMDA-R; furthermore, the activation of NMDA-R increases oxidative stress and calcium load in the mitochondria, leading to cell death (5).

Matrix metalloproteinases (MMPs) are the Zn-containing endopeptidase. The MMPs are involved in extracellular matrix remodeling, leading to arrhythmogenesis (7, 17, 23). MMP activation in HHCY causes endothelium-myocytes uncoupling and is arrhythmogenic (19, 21, 22). NMDA-R antagonist inhibits MMP activation (14) and attenuates SCD (12).

Intracellular localization of MMP has been suggested (13). MMP-2 is synthesized by both cardiac myocytes and fibroblasts and is colocalized with contractile proteins such as troponin I within myofilaments (24), leading to the decline in contractile performance. Others and we have shown the presence of MMP in the mitochondria (mtMMP) (10, 11, 15). The abnormal activation of MMP impairs the mitochondrial function (25). Very recently, by using a pharmacological blocker for NMDA-R1, our laboratory has suggested that HCY agonizes NMDA-R1 and causes a decline in myocyte contractility, in part, by inducing MMP-9 in the mitochondria (16). However, these studies did not reflect the specific contribution of cardiomyocyte NMDA-R1 on contractile function in the setting of HHCY. Therefore, in the present study, the mechanistic role of cardiac NMDA-R1 on the myocyte contractile function was determined by the generation of cardiomyocyte-specific deletion of NMDA-R1.

MATERIALS AND METHODS

Generation of cardiac-specific knockout of NMDA-R1 gene.

Breeding pairs of heterozygous mice with floxed targeted NR1 gene [B6.129S4-Grin1tm2Stl/J (NR1fl/fl)] and heterozygous mice with α-myosin heavy chain (MHC) Cre transgene [B6.129-Tg (Myh6-cre/Esr1)1Jmk/J (α-MHC-Cre)] were procured from The Jackson Laboratory (Bar Harbor, ME). The mice heterozygous for floxed NMDA-R1 allele (NR1fl/+) were bred to homozygosity (NR1fl/fl) with no birth defects and were healthy until adulthood (4 wk). These were then bred with α-MHC Cre transgenic mice to generate the mice homozygous for floxed NMDA-R1 allele, carrying the hemizygous α-MHC Cre transgene (NR1fl/fl; Cre+). This breeding also produced the following littermates: NR1fl/fl, NR1fl/+, and Cre+ (genetic controls) . At 5 wk of age, mice from the four genotypes were given tamoxifen intraperitoneally (20 mg/kg body wt, dissolved at the final concentration of 4 mg/ml in 30% of ethanol-sterile PBS) or vehicle daily for 5 days. To demonstrate the Cre-directed excision of NR1 gene, Western blot and confocal imaging were conducted for NMDA-R1 subunit in the isolated cardiomyocytes. The protocol for the induction and measurement of HHCY was described elsewhere (16). Briefly, HHCY was induced in the mice by administering 1.8 g of DL-HCY/l in the drinking water for 10 wk. The following treatment groups used were: wild type (WT), WT + HCY, NR1fl/fl/Cre + HCY, NR1fl/fl/Cre, and the genetic control for NR1fl/fl/Cre (NR1fl/fl). Animal experiments were carried out according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Louisville.

Adult ventricular myocyte isolation.

Single ventricular myocytes from the adult mice heart were isolated according to the protocol as described elsewhere (16). Briefly, hearts from 8- to 10-wk-old mice were removed rapidly and perfused with calcium-free perfusion buffer (in mmol/l: 120.4 NaCl, 14.7 KCl, 1.2 MgSO4·H2O, 0.6 Na2HPO4, 0.6 KH2PO4, 10 Na-HEPES, 4.6 NaHCO3, 30 taurine, 10 glucose, and 10 butanedione monoxime, pH 7.4) and then with the same buffer with added Liberase Bledzyme 4 (0.9 mg/ml) (Roche Applied Science, Indianapolis, IN) for 15–20 min. Ventricles were removed and resuspended in perfusion buffer with 10% serum, and 1.25 μmol/l calcium was added to stop the digestion. Calcium was reintroduced into cells and was maintained at room temperature in Hanks' buffer (5.6 mmol/l d-glucose and 1.25 μmol/l calcium). Cell yield was ∼50–70% with ∼75–80% viability. There was no notable difference of yield between WT and cardiac-specific NMDA-R1 knockout (KO) mice.

Immunoblotting.

Western blots were carried out using a standard protocol as described elsewhere (16) using antibodies specific to NMDA-R1. The blots were immunodetected using appropriate horseradish peroxidase-conjugated secondary antibodies with the enhanced chemiluminescence plus detection kit. The mitochondrial purity was determined by probing the cytoplasmic and mitochondrial fraction with anti-prohibitin (mitochondrial marker) and anti-glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic marker).

Confocal microscopy.

Isolated cardiomyocytes were processed for immunoconfocal staining using the protocol described elsewhere (16). The images were acquired using a laser confocal microscope (FluoView 1000).

Mitochondrial reactive oxygen species.

Mitochondrial reactive oxygen species (ROS) were detected by loading the cells with mitochondrial-specific, redox-sensitive fluorophore dihydrorhodamine 123 at 5 μmol/l, and the images of 40–50 cells were acquired and quantitated using ImagePro image analysis software.

Mitochondrial nitric oxide levels.

Mitochondrial nitric oxide (NO) levels were determined by selective amperometric oxidation using an NO-sensitive electrode (Apollo 4000; World Precision Instruments) as per the manufacturer's guideline. The NO-sensitive electrode that was selective for NO levels in aqueous solutions was calibrated by generating stoichiometric standards from the reaction as follows: 2KNO2 + 2KI + 2H2SO4·2NO + I2 + 2H2O + 2K2SO4. To measure the mitochondrial NO levels, 1 mg of mitochondrial protein was added to 1 ml of respiration buffer, and the NO levels were detected with a NO-sensitive electrode (amiNO 100; Innovative Instruments, Tampa, FL) using a 7-μm tip.

Assay of mitochondrial permeability transition.

Mitochondrial permeability transition (MPT) was determined using the protocol described elsewhere (16). Briefly, 250 μg of mitochondrial protein were resuspended in the swelling buffer containing (in mmol/l) 250 sucrose, 10 Tris-morpholinosulfonic acid, 0.05 EGTA, pH 7.4, 5 pyruvate, 5 malate, and 1 phosphate, and mitochondrial swelling was determined by a decrease in light absorption at 540 nm. The MPT was measured before and after the addition of CaCl2 (250 μmol/l).

Cell shortening/relengthening.

The contractile property of the adult ventricular myocytes was determined using a video-based edge detection system (IonOptix, Milton, MA) described elsewhere (16). The myocytes were field stimulated at a frequency of 1.0 Hz and connected to a MyoPacer Field Stimulator (IonOptix). Several parameters (%cell shortening, maximal velocities of contraction and relaxation) were recorded and analyzed using Soft-edge software (IonOptix). The rod-shaped cells with clear striations and no sarcolemmal blebbing were included in the experiment.

Intracellular fluorescence measurement of calcium.

Intracellular calcium was determined using a dual-excitation fluorescence photomultiplier system (IonOptix) as described elsewhere (16). Briefly, a separate cohort of myocytes was loaded with fura 2-AM dye, and the fluorescence was monitored and analyzed offline using Soft-edge software (IonOptix).

Statistical analysis.

A number of physiological and contractility measurements were performed from 12–15 cardiomyocytes from 6–8 hearts in each group. Each dataset of biochemical experiments was analyzed using four to six hearts. Data obtained from the same myocyte were used to express results in terms of the percentage change relative to control (before treatment) for pooling for statistical analysis. Experiments using commercial biochemical assays were performed in triplicate. Values were presented as means ± SE. Statistical significance (P < 0.05) was determined by Student's t-test for two groups. One-way or two-way ANOVA was used to compare the respective data with WT, NR1fl/fl/Cre + NR1fl/fl/Cre+, and NR1fl/fl with or without HCY treatment according to the Bonferroni correction.

RESULTS

Generation of cardiomyocyte-specific deletion of NMDA-R1 using cre-mediated recombination.

Cardiomyocyte-specific deletion of NMDA-R1 was confirmed by Western blot and confocal imaging for NMDA-R1 expression (Fig. 1, A and B).

Fig. 1.

Fig. 1.

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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).

HCY induced oxidative and nitrosative stress in the myocyte mitochondria by agonizing NMDA-R1.

The myocyte mitochondrial fraction was isolated and checked for its purity by Western blot analysis. We observed a significant increase in mitochondrial ROS levels in HCY mouse myocytes compared with WT. Interestingly, the HCY-induced increase in mitochondrial ROS was ameliorated in mice having myocyte-specific deletion of NMDA-R1 (Fig. 2A). NO levels were determined in myocyte mitochondria using an NO-sensitive electrode. We observed that HCY induced the generation of mitochondrial NO (mtNO), and the HCY-induced increase in mtNO was attenuated by cardiac-specific deletion of NMDA-R1 (Fig. 2B). These observations suggested that HCY induced the generation of mitochondrial peroxynitrite by increasing the ROS and NO levels in the myocyte mitochondria.

Fig. 2.

Fig. 2.

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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. NR1fl/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 NR1fl/fl/Cre with HCY (#). Data are representative of at least two different experiments (n = 6/group).

HCY induced MPT by agonizing NMDA-R1.

HCY induced the mitochondrial permeability transition. Interestingly, the HCY-induced increase in MPT was attenuated by specific deletion of NMDA-R1 (Fig. 3). This finding suggested that HCY agonized NMDA-R1 and induced MPT.

Fig. 3.

Fig. 3.

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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 = A540max − A540min). 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 NR1fl/fl/Cre with HCY and treatment (#). Data represent two different experiments (n = 6/group).

Cardiac-specific deletion of NMDA-R1 ameliorated the HCY-induced decline in contractile performance and alters calcium transient.

HCY caused a decrease in myocyte contractile performance as indicated by the decrease in the percentage of peak shortening and maximal rate of relaxation and contraction and prolonged the time to 90% peak shortening (TPS-90) and time to 90% relaxation (TR) compared with WT. The HCY-induced decrease in contractile performance and the prolongation of TPS-90 and TR was ameliorated in the mouse with cardiac-specific deletion of NMDA-R1 (Fig. 4). HCY caused a decrease in contraction amplitude with the increase in calcium concentration compared with WT. Moreover, the HCY-induced decrease in contraction amplitude with the increasing concentration of calcium was attenuated in myocytes with cardiac-specific deletion of NMDA-R1 (Fig. 5). The results suggested that cardiac-specific deletion of NMDA-R1 restored the HCY-induced decline in contractile performance.

Fig. 4.

Fig. 4.

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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. NR1fl/fl/Cre with HCY and treatments (#).

Fig. 5.

Fig. 5.

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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.

To define the mechanisms underlying the restoration of cardiomyocyte contractility in cardiomyocyte-specific KO of NMDA-R1 in the setting of HHCY, intracellular calcium handling was analyzed. It was observed that there was no significant change in the baseline fura 2-AM fluorescence intensity (FFI) and change in intracellular calcium transient (ΔFFI). Interestingly, HCY prolonged the recovery of the calcium transient by agonizing NMDA-R1 (Fig. 6).

Fig. 6.

Fig. 6.

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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.

DISCUSSION

In the present study, we demonstrated that cardiomyocyte-specific deletion of NMDA-R1 attenuated the HCY-induced increase in NMDA-R1 expression. HCY caused the increase in ROS and NO levels in the myocyte mitochondria by agonizing NMDA-R1. Cardiac-specific KO of NMDA-R1 ameliorated HCY-induced mitochondrial permeability transition. Cardiac-specific ablation of NMDA-R1 mitigated the HCY-induced decline in contractile performance and the prolongation of TPS-90 and TR. HCY caused a decrease in contraction amplitude with the increasing concentration of calcium. Furthermore, HCY prolonged the recovery of calcium transient by agonizing NMDA-R1. These observations suggested that HCY increased the generation of mitochondrial peroxynitrite (by the increase in ROS and NO levels in the mitochondria) and caused a decline in myocyte mechanics by altering calcium handling, leading to the functional uncoupling of myocyte contraction. A schematic presentation, based on our previous work on MMP-9 (16) and the results obtained from the present study, is shown in Fig. 7.

Fig. 7.

Fig. 7.

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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.

HCY induces fibrosis and endothelial-myocyte disconnection, leading to the decrease in myocardial contractility. Cardiomyocyte expresses NMDA-R, and the activation of NMDA-R increases oxidative stress and calcium load in the mitochondria, leading to cell death (5). However, the direct impact of HCY on the cardiac myocytes and the underlying mechanism for contractile dysfunction are unclear. In addition, the physiological significance of cardiomyocyte NMDA-R and how the activation of NMDA-R alters the myocyte physiology in the setting of HHCY remain to be elucidated. Recently (16) our laboratory has determined the role of myocyte NMDA-R1 and the possible underlying mechanism in modulating the myocyte mechanics in HHCY. In that study, MK-801 i.e., the competitive pharmacological blocker of NMDA-R1, was administered intraperitoneally, and the myocyte contraction was studied in HHCY. However, these observations ruled out the specific contribution of cardiomyocyte NMDA-R1 gene on the myocyte physiology in HHCY. Because the homozygous deletion of NMDA-R1 was embryonic lethal, cardiac-specific KO of NMDA-R1 was generated in the present study, and the underlying mechanism by which myocyte NMDA-R1 regulated the myocyte mechanics in HHCY was determined.

It is known that MMP activation in HHCY increases the deposition of interstitial collagen between endothelium and myocytes and is arrhythmogenic (19, 21, 22). Furthermore, NMDA-R antagonist inhibits MMP activation (14). We and others have reported the presence of MMP in the cardiac mitochondria (mtMMP); however, the functional consequences of the intracellular MMP activation remain obscure (10, 11, 15). Myocyte MMP is shown to be colocalized with contractile proteins, including troponin I within the myofilaments (24), and the generation of peroxinitrite induces MMP-2 activation, leading to myocyte contractile dysfunction (20). In the present study, we have reported that HCY induces the generation of mitochondrial peroxinitrite in the myocyte mitochondria by agonizing the NMDA-R1. We presented the evidence that targeted deletion of NMDA-R1 caused a decrease in HCY-induced MPT in the myocyte mitochondria. This was consistent with our earlier finding that HCY agonized NMDA-R1 and caused MPT by MMP activation (16).

Cardiomyocyte-specific deletion of NMDA-R1 restored the HCY-induced decline in contractile performance, in part, by maintaining MPT. This may be complementary to our recent finding (16) that HCY activates MMP-9 in the myocyte mitochondria-induced MPT, leading to the decrease in myocyte contraction. This decrease in myocyte mechanics by HCY involved the alteration in calcium transients. In the present study, we reported that HCY prolonged the recovery of calcium transient by agonizing NMDA-R1, suggesting that in the HHCY myocyte the rate of clearance of calcium was slowed compared with WT myocytes.

The results from this study suggest that cardiomyocyte-specific deletion of NMDA-R1 mitigated the HCY-induced myocyte mitochondrial ROS and NO levels. HCY induced MPT by agonizing NMDA-R1. Cardiac-specific KO of NMDA-R1 restores the HCY-induced decrease in myocyte contractile performance, in part, by maintaining MPT. Furthermore, HCY prolonged the recovery of calcium transient by agonizing NMDA-R1.

GRANTS

This research was supported by an American Heart Association Postdoctoral Training Grant (award no. 0625579B) (to K. S. Moshal) and National Heart, Lung, and Blood Institute Grants HL-71010, HL-74185 and HL-88012 (to S. C. Tyagi).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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