INTRODUCTION

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HYPERTENSION and other pressure-overload conditions, such as aortic stenosis, result in pathological left ventricular (LV) hypertrophy (LVH). This LVH, a result of adverse cardiac remodeling, is characterized by a combination of myocyte hypertrophy and nonmyocyte cell proliferation with increased deposition of collagen, culminating in increased fibrosis disproportionate to myocyte hypertrophy (39). These changes initially lead to deleterious effects on diastolic function (due to increased myocardial stiffness) and subsequently to depressed contractile function. In addition, coronary arteriolar remodeling with medial thickening and perivascular fibrosis also accompanies adverse cardiac remodeling in hypertension and can contribute to myocyte loss and myocardial dysfunction (21, 27).

The role of hyperhomocysteinemia (Hhe) in the pathogenesis of cardiovascular disease (CVD) has been the subject of active epidemiological investigation. Cross-sectional, retrospective case-control studies and prospective studies have shown an association between Hhe and CVD, including myocardial infarction and stroke. Several pathophysiological mechanisms have been postulated to explain the link between Hhe and atherothrombotic CVD, including endothelial dysfunction, endothelial cytotoxicity, lipid peroxidation, smooth muscle cell proliferation, increased collagen production, and platelet aggregation (18, 24). In addition to these vascular effects, Hhe has been associated with clinical LVH. A study by Blacher and co-workers (1) examined the relationship between high homocysteine levels and LVH in patients with end-stage renal disease. The results of this retrospective study suggested that there is a positive correlation between LV mass index and plasma homocysteine, even after adjustment for age, sex, systolic blood pressure, and hematocrit.

In light of the prevailing hypotheses regarding macrovascular pathogenic mechanisms of Hhe, it seems possible that Hhe may also act to exacerbate the myocardial and coronary arteriolar remodeling and myocardial collagen deposition caused by hypertension. These structural changes would result in adverse effects on cardiac function and potentially contribute to increased cardiovascular morbidity and mortality. Our experiments, which examined the effects of high homocysteine levels on cardiac structure and function in spontaneously hypertensive rats (SHRs), demonstrated that Hhe causes adverse remodeling of the myocardium and coronary arterioles in hypertension, leading to diastolic dysfunction.

	MATERIALS AND METHODS

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Animals. All procedures in this study were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Three-month-old male SHRs (300-325 g) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and maintained in our institutional Division of Laboratory Animal Medicine facility on a 12:12-h light-dark cycle with free access to chow and water. After 7-10 days of acclimatization to this facility, the animals were randomized into three groups.

Langendorff-perfused hearts. This procedure is routinely used in our laboratories (14). Hearts were isolated from rats in each of the treatment groups after 10 wk of dietary intervention and perfused via the aorta with an oxygenated Krebs-Henseleit solution (37°C) of the following composition (in mM): 118.0 NaCl, 27.1 NaHCO₃, 3.7 KCl, 1.8 CaCl₂, 1.2 MgCl₂, 1.0 KH₂PO₄, and 11.1 glucose. The flow rate was set at 7.0 ml · g heart¹ · min¹, a value similar to that observed when flow is examined at a constant pressure of 80 mmHg; coronary pressure was monitored continuously by a Statham pressure transducer. Both atria were removed, and the ventricles were paced electrically at 250 beats/min by platinum contact electrodes positioned on the interventricular septum. A fluid-filled balloon catheter (connected to a pressure transducer) was placed in the LV to measure intraventricular pressure under isovolumic conditions, and the heart was enclosed in a humidified, temperature-controlled chamber. Contractile function was monitored by measuring the peak pressure and maximum rates of contraction and relaxation (+dP/dt_max and dP/dt_max) at various preload balloon volumes (10-80 µl, a range that elicited maximum contractility in all preparations). These balloon volumes represent the volume of fluid within the balloon and do not include the volume of the balloon itself, which varied very slightly from 48 to 52 µl. In addition to the polygraph recording, all data were digitized and analyzed with the use of acquisition and analysis software (CODAS, DataQ Instruments; Akron, OH).

Morphometric measurement of collagen volume fraction and coronary arteriolar remodeling. Morphometric measurements were done as previously described (29). Sections (5 µm) of formalin-fixed, paraffin-embedded tissue were stained with picrosirius red to identify fibrillar collagen (13). Digital images of stained sections were acquired with a charge-coupled device camera (Hamamatsu; Bridgewater, NJ) attached to a Zeiss Axioskop microscope (Zeiss; Thornwood, NY). The integrated image analysis software packages Axiovision 1 and KS300 version 3.0 (Zeiss) were used for morphometric analysis. Perivascular collagen was measured in 50- to 200-µm vessels (10-15 vessels/section) and expressed relative to the vessel luminal area. Interstitial collagen was analyzed in 30-40 microscopic fields/section, and the interstitial collagen volume fraction was calculated as the percentage of collagen-stained interstitial regions relative to the total ventricular area. The arteriolar wall-to-lumen ratio was analyzed in 50- to 200-µm vessels (10-15 vessels/section).

Hydroxyproline assay. Hydroxyproline was assayed using a procedure described by others (5, 31). The LV apex was cut into small pieces and dried to constant weight in an oven set at 65°C. The samples were then allowed to cool to room temperature, and, after being weighed, the dried tissues were transferred into 1.5-ml Eppendorf tubes. HCl (6 N, 200 µl) was added to each tube to hydrolyze the sample by overnight incubation at 105°C. After the tubes cooled, 200 µl of 6 N NaOH were added to each tube, followed by 600 µl isopropanol, and the samples were mixed well. An equal amount of each sample (10 µl) was aliquoted from the aqueous layer into individual borosilicate glass tubes and diluted with acetate-citrate-isopropanol buffer to a final volume of 400 µl; 100 µl of fresh oxidant solution were added, and the samples were mixed and allowed a 5-min incubation period. Ehrlich's reagent solution (1.3 ml) was added, and the samples were mixed and incubated at 60°C for 30 min. After samples were cooled, the absorbance was measured at 558 nm against a blank. The reagent blank was prepared by substituting 60 µl of buffer for the sample in the reaction mixture. Hydroxyproline concentration, expressed as micrograms per milligram of heart weight, was calculated from the absorbance reading of each sample with the use of a calibration curve prepared by assaying standard solutions.

Immunostaining for -smooth muscle actin. Heart tissue was fixed in formalin and embedded in paraffin. Sections (5 µm) were prepared, and immunohistochemistry was performed using anti-smooth muscle actin (Dako; Carpinteria, CA) with appropriate controls. The image analysis system discussed above was used for morphometric analysis.

Estimation of mast cell number. Sections of heart were stained with toluidine blue to identify mast cells by metachromasia (26). A pathologist counted the total number of mast cells in each section in a blinded fashion.

Statistical analysis. Data were evaluated by ANOVA with a Student-Newman-Keuls post hoc test using SigmaStat (SPSS; Chicago, IL). The criterion for significance was a P value of <0.05. Data are reported as means ± SE.

	RESULTS

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We used the SHR model of genetic hypertension to determine whether short-term Hhe exacerbates myocardial and coronary arteriolar remodeling and alters cardiac function in the hypertensive heart.

Animal model of Hhe. Table 2 shows parameter values for the three groups after 10 wk of treatment with the amino acid-defined diets detailed in Table 1. Clinically, Hhe has been classified as moderate (15-30 µmol/l), intermediate (30-100 µmol/l), or severe (>100 µmol/l), with normal values considered to be <15 µmol/l (9). Mean plasma homocysteine levels in our animal models were in the normal (control), IH, and SH range. In fact, homocysteine levels in the SH group were very high (200 µmol/l) but not beyond those observed in certain inborn errors of metabolism (19). Body weight and heart weight were reduced in rats fed the SH diet compared with the same parameters in the control and IH groups; however, the ratio of heart weight to body weight did not differ significantly among groups. Chow intake was reduced in the SH group when compared with the other two groups (data not shown). Diastolic and systolic blood pressure and pulse pressure tended to increase in the IH group and decrease in the SH group; however, no significant differences were observed in these parameters.

Effects of IH and SH on basal LV function. Isolated Langendorff-perfused heart preparations were used to examine effects of Hhe on LV function. After the heart had stabilized under in vitro conditions, the volume in the LV balloon was increased in 10-µl increments, and intraventricular pressure was monitored for diastolic pressure, peak systolic pressure, +dP/dt_max and dP/dt_max. As shown in Fig. 1A, there was a shift of the pressure-volume curve leftward and upward with Hhe; i.e., greater elevations in diastolic pressure were observed at given balloon volumes, with changes reaching statistical significance in the SH group (as compared by ANOVA and post hoc analysis). Because diastolic pressure was generally not detectable at balloon volumes <30 µl, data shown in Fig. 1A include only those values obtained at volumes 30 µl. Hearts from IH rats tended to have a greater diastolic pressure than those from controls at balloon volumes of 70 and 80 µl; however, there was no significant difference between the IH and control groups or between the IH and SH groups.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of balloon volume on diastolic left intraventricular (LV) pressure (A), left intraventricular developed pressure (peak systolic  diastolic; B) and the relationship between left intraventricular diastolic and developed pressures (C) in Langendorff-perfused hearts isolated from control (; n = 8), intermediate hyperhomocysteinemic (IH; ; n = 9), and severe hyperhomocysteinemic (SH; ; n = 9) male spontaneously hypertensive rats (SHRs). Hearts were perfused with oxygenated Krebs-Henseleit buffer (37°C) at a flow rate of 7.0 ml · g tissue1 · min1 and paced at 250 beats/min. The balloon volume was increased in 10-µl increments, with values being recorded after steady state was achieved. A: values for diastolic pressure were not detectable at balloon volumes <30 µl. The SH group was significantly different from controls (ANOVA, P < 0.05). B and C: values for developed pressure are presented as a percentage of the maximum observed value (see text). Vertical and horizontal bars represent means ± SE.

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Effects of IH and SH on cardiac remodeling. We examined the effects of elevated homocysteine levels on the myocyte and nonmyocyte compartments in hypertensive hearts. Mean myocyte diameter, measured at the level of the nucleus, did not vary significantly among groups (data not shown). Perivascular collagen deposition around 50- to 200-µm vessels (~10-15 vessels/section) was measured in picrosirius red-stained sections and normalized to the luminal area. As shown in Table 3, perivascular collagen was increased significantly in the IH and SH groups compared with controls. Figure 2, A-C, shows representative picrosirius red-stained sections from the three groups that demonstrate the enhanced perivascular collagen deposition. Interstitial collagen (measured in 30-35 fields/section) also tended to increase in the IH and SH groups (Fig. 2, D-F); however, significant differences were not observed (Table 3). We also measured the hydroxyproline content in LV myocardium as a marker of collagen concentration. As shown in Fig. 3, tissue hydroxyproline levels increased with Hhe (control: 3.1 ± 0.52 µg/mg, IH: 4.3 ± 0.19 µg/mg, and SH: 5.27 ± 0.43 µg/mg). Figure 2, G-I, shows representative vessels from each group after immunostaining for -smooth muscle actin; increasing medial thickness of the vessels was observed with increasing levels of plasma homocysteine. As shown in Table 3, the wall-to-lumen ratio tended to increase in both Hhe groups, reaching statistical significance in the SH group.

View larger version (109K): [in this window] [in a new window]   Fig. 2.   Representative stained heart sections. Picrosirius red-stained sections from control (A), IH (B), and SH (C) groups show hyperhomocysteinemia (Hhe)-induced perivascular collagen accumulation and arteriolar wall thickening. Similarly stained sections from the control (D), IH (E), and SH (F) groups show a trend toward increases in interstitial collagen in the IH and SH groups when compared with controls. -Smooth muscle actin immunostaining shows Hhe-induced medial thickening when comparing representative vessels from the control (G), IH (H), and SH (I) groups. Toluidine blue-stained sections of the LV from control (J), IH (K), and SH (L) groups show an increased number of mast cells with Hhe. Arrows point to mast cells. See Table 3 for total mast cell numbers per section. Original magnification was ×200 for A-F and ×100 for G-L.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Hydroxyproline content of LV myocardium isolated from control, IH, and SH groups. Data show increases in the SH group compared with controls (P = 0.003). Vertical bars represent SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We examined a novel link between Hhe and the burden of CVD. Because hypertension and Hhe are highly prevalent in the general population, the potentiation of adverse cardiac remodeling in hypertensive hearts by Hhe would be of great clinical importance. Our results indicate that IH and SH increase collagen levels in the hypertensive LV, cause coronary arteriolar wall thickening, and result in diastolic dysfunction.

Hhe has been linked to CVD in a number of previous reports. McCully (19) reported that SH resulting from inborn errors of metabolism results in vascular disease, and the prevalence of elevated plasma homocysteine levels increases from an estimated 5% in the general population to 13-47% in patients with symptomatic vascular disease (24). Retrospective and prospective studies have also shown an association between Hhe and CVD, including myocardial infarction and stroke (3, 23, 28). An interesting small clinical study by Blacher and coworkers (1), in which LV mass and plasma homocysteine levels were measured in 75 patients with end-stage renal disease undergoing hemodialysis, showed a highly significant positive correlation between echocardiographically measured LV mass index and plasma homocysteine, even after adjustment for age, sex, systolic blood pressure, and hematocrit. These data suggest that there is a relationship between Hhe and LVH.

Cardiomyocytes account for approximately one-third of the cells in the myocardium, whereas they occupy 75% of its volume (10, 41). A fibrillar collagen network forms the scaffolding that leads to coordinated myocyte contraction and myocardial force generation (39). Hypertension and other pressure-overload conditions, such as aortic stenosis, are characterized by a combination of myocyte hypertrophy and nonmyocyte cell proliferation with increased levels of collagen. The resultant pathological LVH is characterized by increased fibrosis disproportionate to myocyte hypertrophy (39). This leads initially to deleterious effects on diastolic function and subsequently to depressed systolic function due to interference with coordinated myocyte contraction. Hence, the amount of collagen in the myocardium seems to be a major determinant of the development of cardiac dysfunction in hypertension. The development of heart failure in the SHR model is characterized by increased expression of the precursors of types I and III collagen during the transition from stable cardiac hypertrophy to overt heart failure (2). In addition, Varo and coworkers (37) demonstrated that pathological LVH with myocardial fibrosis in the SHR model is characterized by increased LV collagen concentration, increased steady-state mRNA levels for tissue inhibitor of metalloproteinase-1 (TIMP-1), which regulates collagen degradation, and decreased activity of collagenase [matrix metalloproteinase-1 (MMP-1)], the rate-limiting step in extracellular collagen degradation (16, 40).

Structural analysis of the heart in patients with hypertension has also revealed changes in the coronary arterioles. Munhdenke and co-workers (21) showed that arterioles in patients with hypertensive heart disease have significant increases in medial area and perivascular collagen. They also showed that this remodeling is associated with reduced coronary reserve and clinical evidence of myocardial ischemia in the absence of epicardial coronary disease. In the SHR model that we used, medial thickening and perivascular fibrosis were shown to correlate with the degree of hypertension (11). This progressive cardiac remodeling and fibrosis have been postulated to increase the diffusion distance of oxygen and nutrients for myocytes, thereby contributing to myocyte dysfunction and death. Hence, these changes could lead to progressive myocyte loss, further worsening myocardial function (22, 27).

The human cardiovascular system has been shown to be limited in its ability to metabolize homocysteine. Chen and coworkers (4) determined the activity and protein levels of two major enzymes involved in homocysteine metabolism, cystathionine -synthase and betaine homocysteine methyltransferase, in human heart tissue from rejected donor hearts. The activities of both enzymes were minimal, indicating that the heart is limited in its ability to metabolize homocysteine via these pathways and thus depends primarily on the folate- and vitamin B₁₂-dependent remethylation (methionine synthase) pathway to clear homocysteine. Hence, it is likely that the heart may be especially sensitive to the effects of elevated plasma homocysteine levels.

We examined the effect of Hhe in SHRs using two diets based on homocystine (the disulfide form of homocysteine) supplementation. The diets were successful in elevating plasma homocysteine levels to the intermediate and severe ranges. The SH diet combined deficiencies of choline, methionine, and folate with homocystine supplementation and has been used previously in studies of carotid vascular disease (30). The plasma homocysteine level seen in the SH group in the present study was greater than that documented in the previous report (30), possibly because of the longer duration of dietary treatment. As discussed below, it is possible that folate deficiency played some role in the observed cardiac effects in the SH group; however, there was no significant decrease in hemoglobin or hematocrit after the 10-wk treatment period in our study (data not shown). Nonetheless, because Hhe in the general population frequently coexists with folate, vitamin B₆, or vitamin B₁₂ deficiency, this model has close parallels to the human condition. Blood pressure after the 10 wk of treatment showed a tendency to increase in the IH group [as reported previously (20)] and a tendency to decrease in the SH group. Similarly, as indicated in Fig. 1B, the shift of the curve showing effects of intraventricular balloon volume on LV developed pressure in the IH group was partially reversed in the SH group. The absence of a "concentration dependency" in terms of the effects of homocysteine on blood pressure and the "ventricular function curve" cannot be explained by current data, but it is possibly mediated by folate/methionine/choline deficiency-induced actions in SH that antagonized the effects of Hhe.

The results of our experiments demonstrated that Hhe is associated with a significant increase in perivascular collagen in hypertensive hearts. Interstitial collagen levels also tended to increase in both Hhe groups relative to controls, but this did not reach statistical significance. This parallels the actions of various chemokines known to affect myocardial remodeling (e.g., angiotensin II and aldosterone), which initiate myocardial collagen deposition in the perivascular region followed by an interstitial distribution (39, 40). The interstitial collagen deposition could represent replacement fibrosis after myocyte loss (39). In addition, we demonstrated an increase in the hydroxyproline content of LV myocardium, confirming that total collagen content increased with increasing plasma levels of homocysteine.

The sequence of events leading from elevated plasma homocysteine levels to increased collagen levels is a matter of speculation. Potential mechanisms include possible direct effects of homocysteine on fibroblast proliferation and function leading to increased collagen production or direct effects on collagen metabolism (35). Tyagi and coworkers (36) studied the effect of homocysteine on MMP activity in human coronary artery extracts. They showed a biphasic response, with an increase in collagenolytic activity at homocysteine concentrations <0.1 mM. In addition, homocysteine has been reported to induce TIMP-1 expression in vascular smooth muscle cells (32). Alternatively, high homocysteine levels may cause endothelial dysfunction and expression of adhesion molecules (12) followed by adhesion and activation of inflammatory cells, secretion of chemokines/growth factors, altered fibroblast function, and collagen deposition. The proximate events and signaling mechanisms that influence fibroblast, endothelial, or inflammatory cell function may include oxidative stress or signaling pathways such as protein kinase C and cell cycle proteins (38).

We examined the relation of the inflammatory cell response, specifically mast cell accumulation, to Hhe-induced cardiac remodeling in hypertensive hearts. Mast cells have been correlated with the severity of fibrosis in conditions such as scleroderma, idiopathic pulmonary fibrosis, and keloids (25). Li and co-workers (17) described an association between mast cells and the amount of myocardial fibrosis in transplanted human hearts. Another study (25) examined the relationship of mast cells to cardiac remodeling in the LV of SHRs and found significant increases in perivascular and interstitial collagen levels and an increased number of mast cells compared with normotensive controls. Therapy to decrease blood pressure was associated with a decrease in both collagen accumulation and mast cell number. We observed a dose-dependent increase in mast cells in hypertensive hearts subjected to Hhe that paralleled collagen accumulation. This interesting observation suggests that mast cells may play a role in cardiac remodeling secondary to Hhe in hypertensive hearts.

Hhe also promoted diastolic dysfunction in hearts of hypertensive rats. The pressure-volume relationship was altered, showing an upward/leftward shift with increasing levels of homocysteine. Data from echocardiographic studies have shown that the LV chamber volume is decreased slightly in hearts from SH rats compared with control rats; however, this small change (diastolic diameter was decreased by ~8%, data not shown) cannot account for the observed changes in the pressure-volume curve. In contrast to the effect of Hhe on compliance, the rate of relaxation expressed as dP/dt_max did not differ significantly among the groups. Diastolic dysfunction can result from increased passive chamber stiffness or impaired myocardial relaxation (6). Because there was no evidence of myocyte hypertrophy or impaired myocardial relaxation, it seems likely that the Hhe-induced diastolic dysfunction was caused by collagen accumulation and a resulting increase in myocardial stiffness. This increase in stiffness is a very significant finding because diastolic dysfunction accounts for a significant percentage of patients with heart failure (7), including those resulting from hypertensive heart disease.

The effect of Hhe on systolic function was also assessed in isolated perfused heart preparations. The relationship of peak developed LV pressure to LV balloon volume was shifted leftward with Hhe in the IH group; however, there was no change in the maximal pressure developed. This suggests that the shift in the curve was secondary to increased myocardial stiffness, which resulted in a given increase in volume, resulting in a greater increase in sarcomere length of the cardiomyocytes. These results suggest that the Hhe-induced increase in myocardial stiffness leads to a shift in the ventricular function curve without depression of the maximum achievable contractile response.

In summary, the results of this study suggest a novel mechanism for the contribution of Hhe to cardiovascular morbidity and mortality. Data from the SHR model indicate that the interaction of hypertension and Hhe leads to impaired diastolic function due to potentiation of the adverse myocardial and coronary vascular remodeling in hypertensive hearts. Further studies are required to explore the mechanisms of collagen accumulation and myocardial remodeling as well as the effects of interventions that decrease plasma homocysteine levels.