Homoarginine levels after supplementation by oral gavage
Baseline HA levels without any supplementation were around 0.9 mg/L. After two weeks, plasma levels of animals treated with 200 mg·kg-1·day-1 or 400 mg·kg-1·day-1 HA increased up to 125 mg/L or 274 mg/L, respectively. A standardized protocol for feeding with oral gavage ensured identical food intake to all animals.
Animal data revealed signs of cardiac decompensation and hypertrophy in AB animals
Male Wistar rats with a weight of 117.60 ± 2.04 g were used. As expected, AB was significantly associated with myocardial hypertrophy because of pressure overload. Hence, HW to BW ratio (mg/g), HW to TL ratio (mg/mm), LV to BW ratio (mg/g), LV to TL ratio (mg/mm) as well as RV to TL ratio (mg/mm) were significantly higher (p<0.05) in AB rats compared to SH rats. HA treatment combined with heart failure medication or L-NAME supplementation led to a suppressed hypertrophic response as evidenced by a decline in heart weight (RV + LV) in AB subgroups (p<0.05), whereas solely HA treated AB animals showed tendentially higher values. Animals with AB showed higher lung weights compared to animals without intervention (p<0.05) which may be regarded as an indicator for fluid retention after aortic constriction. Interestingly, AB animals under supplementation with the maximal dose of 800 mg·kg-1·day-1 HA or concomitantly applied heart failure or L-NAME medication showed a significant lower lung weight than their placebo treated AB controls. All animal data are shown in Table 1 and 2.
Homoarginine improves myocardial function and lowers blood pressure as determined by echocardiographic and invasive hemodynamic measurements
Pressure overload led to an enlargement of LV chamber as evidenced by increased LVEDD, aggravated systolic function with increased LVESD, reduced EF and fractional shortening (FS), and LV hypertrophy (Table 4). Within the AB group, echocardiographic measurements demonstrated a dose-dependent increase of LV systolic function in HA treated animals compared to placebo treated animals (Table 3, 4). EF was enhanced after HA supplementation with significant results under solitary AB 800 mg·kg-1·day-1 HA treatment and a combination therapy of HA with spironolactone or lisinopril (48 ± 3 [AB 0 mg·kg-1·day-1 HA] vs. 57 ± 3% [AB 800 mg·kg-1·day-1 HA], 60 ± 3% [AB 800 mg·kg-1·day-1 HA w/ spironolactone] and 60 ± 4% [AB 800 mg·kg-1·day-1 HA w/ lisinopril], p<0.05) (Figure 2). Similarly, FS was markedly enhanced in AB animals treated with either 800 mg·kg-1·day-1 HA alone or in combination with spironolactone or lisinopril compared to untreated AB animals (20 ± 2 [AB 0 mg·kg-1·day-1 HA] vs. 25 ± 2% [AB 800 mg·kg-1·day-1 HA], 27 ± 2% [AB 800 mg·kg-1·day-1 HA w/ spironolactone] and 27 ± 2% [AB 800 mg·kg-1·day-1 HA w/ lisinopril], p<0.05). AB rats given doses of 200, 400 mg·kg-1·day-1 or 800 mg·kg-1·day-1 HA w/ L-NAME also exhibited a rise of EF and FS, albeit without reaching statistical significance in comparison to untreated AB animals. LVEDD and LVESD dimensions were enlarged in AB animals in comparison to SH animals, whereas we observed no significant changes within AB or SH rats (p<0.05) (Figure 2).
It should be noted that echocardiographic measurements were performed under varying heart rate, especially elevated in AB animals under HA and heart failure medication (463 ± 25 [AB 0 mg·kg-1·day-1 HA] vs. 535 ± 11 mmHg [AB 800 mg·kg-1·day-1 HA w/ spironolactone] and 554 ± 18 mmHg [AB 800 mg·kg-1·day-1 HA w/ lisinopril], p<0.05). Therefore, parameters of LV function could be underestimated. SH operated animals demonstrated no significant changes of EF or FS after HA treatment (Table 3, 4).
Invasive pressure measurements confirmed effectiveness of AB and showed sufficient peak-to-peak gradients compared to SH animals (6 ± 5 [SH 0 mg·kg-1·day-1 HA] vs. 94 ± 15 mmHg [AB 800 mg·kg-1·day-1 HA], p<0.05) (Table 5, 6; Figure 3). Peak-to-peak pressure gradients did not differ significantly among the different subgroups of animals undergoing AB compared to controls. Also, values of end-systolic LV pressure were increased after AB (124 ± 7 [SH 0 mg·kg-1·day-1 HA] vs. 188 ± 13 mmHg [AB 800 mg·kg-1·day-1 HA] and 194 ± 11 mmHg [AB 800 mg·kg-1·day-1 HA w/ spironolactone], p<0.05). A reduction of arterial pressure was detected in both, the SH and AB groups, upon 800 mg·kg-1·day-1 HA treatment alone or if combining with heart failure medication (118 ± 7 [SH 0 mg·kg-1·day-1 HA] vs. 99 ± 3 mmHg [SH 800 mg·kg-1·day-1 HA w/ spironolactone], p<0.05; 108 ± 3 [AB 0 mg·kg-1·day-1 HA] vs. 91 ± 3 mmHg [AB 800 mg·kg-1·day-1 HA w/ lisinopril], p<0.05).
Histopathology: Homoarginine attenuates myocardial hypertrophy and fibrosis in AB animals
Myocardial dysfunction in AB rats was associated with myocardial hypertrophy and morphological characteristics of adverse remodeling. The HA treatment after induction of AB has led to several beneficial effects.
First, we evaluated the effect of HA treatment on cardiac myocyte size (as shown by H&E staining). Four weeks following AB operation, cardiomyocyte cross-sectional area was expectably increased across all AB subgroups compared to SH animals (Table 7, 8; Figure 4). Cardiac hypertrophy was attenuated by HA treatment and even more with each applied comedication. Myocytes from AB animals, either treated with HA alone or in combination with spironolactone, lisinopril or L-NAME, were significantly smaller in comparison to AB rats with placebo. AB animals with 800 mg·kg-1·day-1 HA w/ lisinopril presented the most significant decrease with approximately 22% smaller myocyte size than placebo treated AB animals (142 ± 2.0 [AB 800 mg·kg-1·day-1 HA w/ lisinopril] vs. 183 ± 2.4 RU [AB 0 mg·kg-1·day-1 HA], p<0.05). Solitary treatment with 800 mg·kg-1·day-1 HA resulted in a reduction of myocyte cross-sectional area by approximately 10% compared with untreated AB rats (164 ± 1.7 [AB 800 mg·kg-1·day-1 HA] vs. 183 ± 2.4 RU [AB 0 mg·kg-1·day-1 HA], p<0.05). Among SH operated animals, groups with spironolactone, lisinopril and L-NAME treatment revealed smaller cardiomyocytes.
Quantitative evaluation of volume percent collagen (Table 9, 10; Figure 5) revealed a significant increase of collagen deposition in the interstitium and around vascular tissue (expressed as collagen area fraction in percent) after AB compared with SH operated animals. Within the AB group, myocardium of animals treated with either HA alone or combined with spironolactone, lisinopril or L-NAME demonstrated a marked reduction of fibrotic areas as shown by sirius red staining. AB animals experienced a clearly dose-dependent reduction in myocardial fibrosis with the most significant decrease of approximately 59% among animals treated with 800 mg·kg-1·day-1 HA w/ L-NAME (collagen area fraction: 5.08 ± 0.36 [AB 0 mg·kg-1·day-1 HA] vs. 2.09 ± 0.21% [AB 800 mg·kg-1·day-1 HA w/ L-NAME], p<0.05).
To summarize, AB animals showed increased interstitial fibrosis with extensive collagen deposition, enlarged cardiomyocytes, and sporadically spots of myofiber disarray in ventricular myocytes. Treatment of AB animals with HA, either alone or combined, resulted in a dose-dependent decline in myocyte size and cardiac fibrosis.
Molecular markers of hypertrophy and fibrosis are downregulated after homoarginine treatment
In AB animals, doses of HA were inversely associated with the molecular hypertrophic markers ANF, BNP and β-MHC. Especially in the analysis of β-MHC and ANF, rats within the 800 mg·kg-1·day-1 AB group experienced a marked downregulation in comparison to unfed AB animals. β-MHC was downregulated by 32% (p=0.29), ANF by 45% (p=0.15) and BNP by 24% (p=0.25). Combination of HA with spironolactone, lisinopril or L-NAME resulted in more pronounced reductions. Herein, animals following HA w/ lisinopril treatment were found to exhibit the greatest reduction of ANF by 78% (p=0.04) and β-MHC by 42% (p=0.19), whereas BNP was less downregulated by 28% (p=0.22) (Figure 4). Furthermore, mRNA levels of col5a1, a molecular marker of fibrosis, were downregulated in the AB group according to rising doses of HA (downregulation by 64% in the 800 mg·kg-1·day-1 AB group compared to the AB control group with placebo treatment, p=0.06). AB animals within the HA w/ spironolactone treatment group experienced a downregulation of 73% (p=0.06) in comparison to AB animals with placebo treatment (Figure 5).
SH operated animals demonstrated nothing more than a trend towards lower values of molecular gene expression in some subgroups with the most obvious results using BNP and col5a1 (Figure 4, 5). Furthermore, in a small subanalysis of SH and AB animals (n=14) the inflammatory markers IL17A and TNFSF14 were found to be downregulated after HA treatment (Figure 6).
Isolated cardiac fibroblast mRNA responses to treatment with homoarginine
Col5a1 mRNA expression levels showed a significant concentration-dependent downregulation in cardiac rat fibroblasts upon HA treatment (p<0.05). Results are illustrated in Figure 5.
Adverse side effects and mortality
Finally, we explored potential negative side effects of HA treatment on behavior, wound healing, growth, and cardiac function. Drug-treated animals showed no behavioral disorders compared to their placebo groups. Moreover, we did not observe an increased proneness for wound infections or growth-promoting effects on body weight. HA treatment did neither result in a deterioration of heart function nor in any other serious side effect (Table 1, 2). No deaths due to HA treatment were observed. Peri- and postoperative mortality was approximately 30% in the banding group and <10% in the SH operated group. No difference was found in mortality between groups after drug treatment with different doses of HA (e. g. AB control group 64% vs. AB groups with HA supplementation 66%, p>0.05). Postmortem analysis revealed overt signs of heart failure (enlargement of the left and right ventricle, pleural effusion) in all AB operated rats that died spontaneously.