In this study, we evaluated the effect of chronic 12- and 56-wk-long treatments with TMAO on the development of hypertension and its complications in spontaneously hypertensive rats (SHRs).

Hypertension is a major risk factor of cardiovascular events. Recently, it has been found that high plasma level of trimethylamine oxide (TMAO), a liver metabolite of gut bacteria-produced trimethylamine (TMA), is associated with an increased cardiovascular risk ( 18 , 30 , 35 , 36 , 42 , 44 ). However, the mechanism of the plasma TMAO increase and effect of TMAO on the circulatory system are not clear. On the one hand, several experimental studies have suggested that TMAO may produce harmful changes in the circulatory system, including platelet hyperreactivity ( 56 ), decreased β-oxidation of fatty acids in myocardiocytes ( 21 ), alterations in cholesterol and sterol metabolism ( 42 ), exacerbated inflammatory reactions of vascular wall, and increased reactive oxygen species production ( 41 ). In addition, we previously found that a 2-wk 100-fold increase in blood TMAO in normotensive rats prolonged the hypertensive effect of concomitantly given low-dose angiotensin II; however, it did not produce significant effects if given alone ( 45 ). On the other hand, manifold higher TMAO plasma levels were observed in humans after ingestion of fish and vegetarian diet than after ingestion of red meat and eggs ( 4 ). Therefore, it seems that fish-rich and vegetarian diet, which is beneficial or at least neutral for cardiovascular risk, is associated with a significantly higher plasma TMAO than diets rich in red meat and eggs, which are considered to increase the cardiovascular risk ( 1 , 16 ). Moreover, TMAO plays a protective role in deep sea animals that are exposed to high hydrostatic pressure. Namely, deep sea fishes use TMAO as a piezolyte, a molecule that counteracts the protein-destabilizing effects of hydrostatic pressures ( 47 , 50 ). Finally, TMAO has been found to protect proteins from the destabilizing effects of urea, high temperature, and NaCl ( 46 ), and, as a protein stabilizer, it has been tested to treat protein-folding diseases ( 3 , 40 , 43 , 53 ).

Differences between the groups were evaluated by one-way ANOVA followed by a Tukey’s post hoc test or by t -test when appropriate. The Kolmogorov-Smirnov test was used to test normality of the distribution. A value of two-sided P < 0.05 was considered significant. Analyses were conducted using Dell Statistica (version 13, Dell, Tulsa, OK).

In the acute hemodynamic experiments, MABP, diastolic blood pressure (DBP), systolic blood pressure (SBP), HR, and LVEDP were calculated on the blood pressure tracing by AcqKnowledge 4.3.1 Biopac software (Biopac Systems). To evaluate ECG, lead II was used.

In the chronic telemetry experiments, MABP and HR were calculated by ART software (Data Sciences). For the evaluation of MABP and HR changes over time, one-way ANOVA for repeated measures followed by Tukey’s post hoc test were used. Differences between the groups were evaluated by multivariate ANOVA followed by a Tukey’s post hoc test.

Sixty-week-old rats in the WKY ( n = 6), SHR-Water ( n = 8), and SHR-TMAO ( n = 8) groups were maintained for 2 days in metabolism cages to evaluate 24-h water and food balance and to collect urine for biochemical analysis. The next day, rats were anesthetized with urethane (1.5 g/kg body wt ip, Sigma-Aldrich) and underwent echocardiography using a Samsung HM70 ultrasound system equipped with a linear probe at 5–13 mHz. The probe was placed on the shaved chest wall to obtain images from the right parasternal short axis. After the echo examination, the left femoral artery and right common carotid artery were cannulated for hemodynamic recordings. LVEDP, ABP, HR, and histopathological and biochemistry tests were performed as described above for 16-wk-old rats.

Gene expression levels were assessed using real-time PCR and were measured with the ABI-Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). Specific probes and primers of TaqMan Gene Expression Assays (ThermoFisher Scientific) for rat angiotensin II receptor type 1a, angiotensin II receptor type 2, angiotensinogen, and GAPDH were applied to each well of the 96-well optical reaction plate (Applied Biosystems). All reactions were run in duplicate, no template control for tested genes in triplicate, and the threshold cycles (C t ) were determined.

Total RNA from tissue was extracted using EZ1 RNA Tissue Mini Kit and Biorobot EZ1 (Qiagen) according to the manufacturer's protocol. RNA was quantified by measuring absorbance at a wavelength of 260 nm using a spectrophotometer NanoDrop 1000. RNA samples were frozen and stored at −80°C for subsequent analysis.

Tissue sections fixed in 10% buffered formalin were dehydrated using graded ethanol and xylene baths and embedded in paraffin wax. Sections of 3–4 μm were stained with hematoxylin and eosin as well as van Gieson stain (for connective tissue fibers). General histopathological examination was evaluated at magnifications of ×10, ×40, and ×100 (objective lens) and ×10 (eyepiece), and photographic documentation was made. Additionally, we performed the following morphometric measurements: width of cardiomyocytes, wall thickness of the aorta, and carotid artery at a magnification of ×40 (objective lens). The level of fibrosis was calculated as a percentage of the connective tissue in the general surface of the myocardium. Micrographs were taken at magnifications of ×40 (objective lens) and ×10 (eyepiece) using a standard light microscope (Olympus BX41) and CellSens software (Olympus, Tokyo, Japan). For each sample, 10 fields of view of the myocardium in histological slides were evaluated. The percentage of fibrosis was calculated using ImageJ software (National Institutes of Health, Bethesda, MD).

The following EIAab Kits (EIAab Science, Wuhan, China) were used for the evaluation: NH 2 -terminal pro-B-type natriuretic peptide (NT-proBNP; catalog no. E0485r), angiotensin II (catalog no. E0005Ge), aldosterone (catalog no. E0911Ge), vasopressin (catalog no. E1139Ge), TNF-α (catalog no. E0133r), and IL-10 (catalog no. E0056r). All procedures were performed according to the standard protocol of ELISA kit operating instructions. The absorbance intensity was measured at 450 nm with the Multiskan microplate reader (ThermoFisher Scientific, Waltham, MA). All experiments were performed in duplicate.

Plasma and urine TMA and TMAO were evaluated using a Waters Acquity Ultra Performance Liquid Chromatograph coupled with a Waters TQ-S triple-quadrupole mass spectrometer. The mass spectrometer was operated in the multiple-reaction monitoring-positive electrospray ionization mode, as previously described ( 13 ). Plasma and urine Na + , K + , creatinine, and urea were analyzed using Cobas 6000 analyzer (Roche Diagnostics, Indianapolis, IN).

The next day, rats were anesthetized with urethane (1.5 g/kg body wt ip, Sigma-Aldrich, St. Louis, MO), and the left femoral artery was cannulated with polyurethane catheter for arterial blood pressure (ABP) and HR recording. Recordings were started 40 min after the induction of anesthesia and 15 min after the arterial catheter was inserted. After 10 min of ABP recordings, a Millar Mikro-Tip SPR-320 (2-Fr) pressure catheter was inserted via the right common carotid artery, and simultaneous left ventricular (LV) end-diastolic pressure (LVEDP) and ABP recordings were performed. The catheter was connected to Millar Transducer PCU-2000 Dual Channel Pressure Control Unit (Millar, Houston, TX) and Biopac MP 150 (Biopac Systems, Goleta, CA). For ECG recordings, standard needle electrodes (Biopac Systems) were used. After hemodynamic recordings, blood from the right ventricle of the heart was taken, and rats were killed by decapitation. The heart, kidneys, and arteries were harvested for further analysis.

Six-week-old rats in the WKY ( n = 6), SHR-Water ( n = 7), and SHR-TMAO ( n = 7) groups were implanted with telemetry transmitters (HD-S10, Data Sciences, St. Paul, MN) under general anesthesia with ketamine (100 mg/ml, Bioketan, Vetoquinol Biowet, Gorzow Wielkopolski Poland) at 100 mg/kg body wt and xylazine (20 mg/ml, Xylapan, Vetoquinol Biowet) at 10 mg/kg body wt ip, as previously described ( 38 ). Continuous recordings of heart rate (HR) and mean arterial blood pressure (MABP) were started with ART software (Data Sciences) 1 wk after the surgery and were performed continuously for 9 wk. Afterward, rats were maintained for 2 days in metabolism cages to evaluate 24-h water and food balance and to collect urine for biochemical analysis.

To choose a dose of TMAO, we did pilot studies with SHRs receiving TMAO for 4 wk at a dose of 1 g/l ( n = 3), 0.333 g/l ( n = 3), and 0.1 g/l ( n = 3). We chose a dose that increased blood TMAO concentration three to five times to mimic possible physiological concentrations and to avoid suprapharmacological blood TMAO concentration.

After being weaned, 4- to 5-wk-old SHRs were maintained on tap water (SHR-Water group) or water containing TMAO (333 mg/l, abcr, Karlsruhe, Germany; SHR-TMAO group). WKY rats were used as normotensive controls to discriminate between age-dependent and hypertension-dependent histopathological changes in SHRs.

The study was performed according to Directive 2010/63 EU on the protection of animals used for scientific purposes and approved by the I Local Bioethical Committee in Warsaw (permission: 100/2016). Male SHRs and normotensive Wistar-Kyoto (WKY) rats were obtained from the Central Laboratory for Experimental Animals, Medical University of Warsaw (Warsaw, Poland). Rats were housed in groups of two to three animals in polypropylene cages with environmental enrichment on a 12:12-h light-dark cycle with temperature at 22–23°C, humidity at 45–55%, and food and water ad libitum.

Fig. 13. Cardiac gene expression of angiotensinogen (ATG) and angiotensin II type 1a and 2 receptors (AT 1a R and AT 2 R, respecitively) in 16-wk-old spontaneously hypertensive rats (SHRs) treated with water (SHR-Water; n = 5) or SHRs treated with trimethylamine oxide in drinking water ( n = 5). C t , threshold cycles. Means ± SE are presented.

There were no significant differences in cardiac gene expression of angiotensinogen and angiotensin II type 1a and 2 receptors between the SHR-Water and SHR-TMAO groups ( Fig. 13 ).

Fig. 12. Histopathological picture of arteries in 60-wk-old rats. Rats were divided into the following groups: Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water group), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO group). A – C : wall of the carotid artery (hematoxylin and eosin stain). D – F : coronary arteries in the heart (hematoxylin and eosin stain). G : wall thickness of the carotid artery (in µm; group means ± SE). WKY group: n = 6, SHR-Water group: n = 7, SHR-TMAO group: n = 7. * P < 0.05 vs. the WKY group (by one-way ANOVA followed by a Tukey’s post hoc test).

In contrast to the WKY group, SHR-Water and SHR-TMAO groups showed hypertrophy of smooth myocytes, thickened connective tissue, and endothelial damage ( Figs. 7 and 12 ). The SHR-Water and SHR-TMAO groups showed a significant increase in the thickness of the carotid artery wall ( Fig. 12 ) and aorta ( Fig. 7 ). Additionally, in coronary and renal arteries, we found hypertrophy of smooth myocytes and perivascular fibrosis ( Fig. 7 ). Pathological changes in the coronary arteries were greater in the SHR-Water group, whereas the changes in large arteries were similar between SHR-Water and SHR-TMAO groups.

Fig. 11. Histopathological pictures of hearts of 60-wk-old rats. Rats were divided into the following groups: Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water group), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO group). A – C : myocardium (hematoxylin and eosin stain). D – F : myocyte cross section (hematoxylin and eosin stain). G – I : myocardium (van Gieson stain for connective tissue fibers). J : width of cardiomyocytes (in µm; group means ± SE). K : percentage of fibrosis (group means ± SE). WKY group: n = 6, SHR-Water group: n = 7, SHR-TMAO group: n = 7. * P < 0.05 vs. the WKY group; † P < 0.05 vs. the SHR-Water group (by one-way ANOVA followed by a Tukey’s post hoc test).

There was an increase in the thickness of the LV wall caused by significant hypertrophy of cardiomyocytes in SHR-TMAO and SHR-Water groups. Morphometric analysis showed a significantly greater width of cardiomyocytes in the SHR-Water group than in the SHR-TMAO group. Similarly, connective tissue hyperplasia in the myocardium was significantly greater in the SHR-Water group than in the SHR-TMAO group ( Fig. 7 and Fig. 11 ).

There were no disturbances in heart rhythm in ECG recordings. SHRs showed significantly wider QRS complexes than WKY rats. There were no significant differences between SHR-Water and SHR-TMAO groups in ECG morphology ( Table 2 and Fig. 10 ).

Fig. 9. Simultaneous recording of arterial blood pressure (ABP; in mmHg) and left ventricular end-diastolic-pressure (LVEDP; in mmHg) in 60-wk-old Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO). A : group means ± SE. LVEDP was calculated as the average of 10 consecutive LVEDP intervals for each rat. WKY group: n = 6, SHR-Water group: n = 7, SHR-TMAO group: n = 6. * P < 0.05 vs. the WKY group; † P < 0.05 vs. the SHR-Water group (by one-way ANOVA followed by a Tukey’s post hoc test). B – D : analog recordings of ABP and LVEDP from one rat from the WKY, SHR-Water, and SHR-TMAO groups, respectively. Arrows show LVEDP intervals.

There were no significant differences between SHRs and WKY rats in basic echocardiographic parameters, including stroke volume and ejection fraction; however, SHRs showed a trend toward an increased interventricular septum diameter ( P = 0.15) and an increased posterior wall diameter of the LV ( P = 0.2; Table 2 and Fig. 8 ).

Anesthetized 60-wk-old SHRs showed significantly higher MABP, SBP, and DBP than WKY rats. There was no significant difference between SHR-Water and SHR-TMAO groups in MABP and SBP, whereas DBP was lower in the SHR-TMAO group ( Table 2 ).

Fig. 7. Histopathological picture of arteries in 60-wk-old rats. Rats were divided into the following groups: Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water group), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO group). A – C : coronary arteries in the heart (van Gieson stain for connective tissue fibers). D – F : arcuate arteries in the kidney (hematoxylin and eosin stain). G – I : wall of the aorta. J : wall thickness of the aorta (in µm). K : thickness of the periarterial connective tissue in the heart (in µm). WKY group: n = 6, SHR-Water group: n = 7, SHR-TMAO group: n = 7. * P < 0.05 vs. the WKY group; † P < 0.05 vs. the SHR-Water group (by one-way ANOVA followed by a Tukey’s post hoc test).

Fig. 6. Histopathological picture of kidneys in 60-wk-old Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO). A – C : parenchyma of the renal cortex (hematoxylin and eosin stain). D – F : parenchyma of the renal cortex (van Gieson stain for connective tissue fibers).

In contrast to the WKY group, SHR-Water and SHR-TMAO groups showed arcuate and lobed artery thickening caused by hyperplasia of smooth myocytes and moderate hyperplasia of the connective tissue accompanied by focal mononuclear cell infiltrates in the parenchyma, single casts in the renal tubules, and weak to moderate glomerular collapse and glomerulosclerosis ( Fig. 6 and Fig. 7 ). There were no evident differences between SHR-TMAO and SHR-Water groups.

There was no significant difference in plasma angiotensin II and aldosterone levels among groups, whereas the plasma vasopressin level was significantly lower in the SHR-TMAO group than in the SHR-Water group ( Table 2 ).

There was no significant difference between the groups in plasma Na + level, but SHRs showed a significantly lower 24-h urine Na + excretion than WKY rats. The SHR-TMAO group showed a moderately higher 24-h urine output and 24-h urine Na + excretion than the SHR-Water group ( Table 2 ).

There was no significant difference between the groups in water intake and urine output; however, SHRs showed a trend towards a higher water intake ( P = 0.2) and 24-h urine output ( P = 0.1) than WKY rats ( Table 2 ).

Fig. 5. Histopathological picture of arteries in 16-wk-old rats. Rats were divided into the following groups: Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water group), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO group). A – C : wall of the carotid artery (hematoxylin and eosin stain). D – F : coronary arteries in the heart (hematoxylin and eosin stain). G : wall thickness of the carotid artery (in µm). WKY group: n = 6, SHR-Water group: n = 6, SHR-TMAO group: n = 6. * P < 0.05 vs. the WKY group (by one-way ANOVA followed by a Tukey’s post hoc test).

In SHRs, we found hypertrophy of smooth myocytes in the coronary arteries and carotid artery, which were more pronounced in the SHR-Water group than in the SHR-TMAO group ( Fig. 5 ). The increase in wall thickness of the aorta was comparable between SHR-Water and SHR-TMAO groups ( Fig. 3, G – I ). In coronary and renal arteries, we found hypertrophy of smooth myocytes and perivascular fibrosis, which were more pronounced in the SHR-Water group than in the SHR-TMAO group ( Fig. 3 ).

Fig. 4. Histopathological picture of heart in 16-wk-old rats. Rats were divided into the following groups: Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water group), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO group). A – C : myocardium (hematoxylin and eosin stain). D – F : myocyte cross section (hematoxylin and eosin stain). G – I : myocardium (van Gieson stain for connective tissue fibers). J : width of cardiomyocytes (in µm; group means ± SE). K : percentage of fibrosis (group means ± SE). WKY group: n = 6, SHR-Water group: n = 6, SHR-TMAO group: n = 6. * P < 0.05 vs. the WKY group; † P < 0.05 vs. the SHR-Water group (by one-way ANOVA followed by a Tukey’s post hoc test).

Morphometric analysis showed cardiomyocyte hypertrophy in SHR-TMAO and SHR-Water groups. The increase in the width of cardiomyocytes in the SHR-Water group was significantly higher than in the SHR-TMAO group ( Fig. 4 ). Additionally, we found connective tissue hyperplasia and perivascular fibrosis in the myocardium in SHRs ( Fig. 3, A – C , and Fig. 4 ). Fibrosis was significantly greater in the SHR-Water group than in the SHR-TMAO group ( Fig. 4 ).

There was no significant difference among the groups in plasma NT-proBNP level; however, the SHR-Water group showed higher plasma NT-proBNP than the SHR-TMAO and WKY groups ( Table 1 ).

Anesthetized 16-wk-old SHRs showed significantly higher MABP and SBP and a trend toward higher DBP ( P = 0.09) compared with WKY rats. There were no significant differences between the SHR-Water and SHR-TMAO groups in MABP, SBP, and DBP ( Table 1 ).

Fig. 3. Histopathological picture of arteries in 16-wk-old rats. Rats were divided into the following groups: Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water group), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO group). A – C : coronary arteries in the heart (van Gieson stain for connective tissue fibers). D – F : arcuate arteries in the kidney (hematoxylin and eosin stain). G – I : wall of the aorta. J : wall thickness of the aorta (in µm). K : thickness of the periarterial connective tissue in the heart (in µm). WKY group: n = 6, SHR-Water group: n = 6, and SHR-TMAO group: n = 6. * P < 0.05 vs. the WKY group; † P < 0.05 vs. the SHR-Water group (by one-way ANOVA followed by a Tukey’s post hoc test).

Fig. 2. Histopathological picture of kidneys in 16-wk-old Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs) treated with water (SHR-Water group), and SHRs treated with trimethylamine oxide in drinking water (SHR-TMAO group). A – C : parenchyma of the renal cortex (hematoxylin and eosin stain). D – F : parenchyma of the renal cortex (van Gieson stain for connective tissue fibers).

There were no evident pathological changes in 16-wk-old rats in the kidney parenchyma ( Fig. 2 ). However, rats in the SHR-Water and SHR-TMAO group exhibited thickening of the tunica media of the lobed and arcuate arteries, which were caused by hypertrophy and hyperplasia of smooth myocytes ( Fig. 3, D – F ).

Rats in the SHR-Water group showed a higher plasma Na + level and a lower 24-h Na + urine excretion than WKY rats. There was no significant difference between SHR-Water and SHR-TMAO groups in those parameters ( Table 1 ).

Rats in the SHR-Water and SHR-TMAO groups showed a higher water intake than WKY rats. There was no significant difference in water intake and urine output between SHR-Water and SHR-TMAO groups; however, the SHR-TMAO group showed moderately lower water intake and urine output compared with the SHR-Water group ( Table 1 ).

Rats in the SHR-Water group showed an insignificantly higher TMAO plasma level compared with WKY rats. The SHR-TMAO group showed significantly higher plasma TMAO compared with the SHR-Water group (4-fold increase). There was no significant difference in plasma TMA levels between groups. However, SHR-TMAO and SHR-Water groups showed significantly lower 24-h urine TMA excretion than the WKY group ( Table 1 ).

There was no significant difference between 16-wk-old rats in the WKY, SHR-Water, and SHR-TMAO groups in body mass. WKY rats showed a higher food intake than SHRs. There were no significant differences in food intake between SHR-Water and SHR-TMAO groups ( Table 1 ).

Fig. 1. Mean arterial blood pressure (MABP; in mmHg; A ) and heart rate (HR; in beats/min; B ) in normotensive Wistar-Kyoto (WKY) rats ( n = 6) and spontaneously hypertensive rats (SHRs) maintained on tap water (SHR-Water group; n = 7) or water containing trimethylamine oxide (SHR-TMAO group; n = 7). Values are means ± SE. * P < 0.05, MABP changes in time in SHRs; † P < 0.05, MABP changes in time in the SHR-TMAO group (by one-way ANOVA for repeated measurements).

Freely moving 7-wk-old SHRs showed a significantly higher MABP than WKY rats. There were no significant differences between 7-wk-old rats in the SHR-Water and SHR-TMAO groups in MABP and HR ( Fig. 1 ). Treatment with TMAO did not affect the development of hypertension in SHRs. Namely, SHR-TMAO and SHR-Water groups showed a significant, gradual increase in MABP, which stabilized in 15- to 16-wk-old rats. This was accompanied by an insignificant decrease in HR. In contrast, WKY rats did not show any significant change in MABP and HR throughout the experiment ( Fig. 1 ).

DISCUSSION

A new finding of our study is that a four- to fivefold increase in plasma TMAO does not exert negative effects on the circulatory system. In contrast, low-dose TMAO treatment is associated with reduced cardiac fibrosis and improved hemodynamic and biochemical parameters of the failing heart in SHRs.

Effect of TMAO on Hypertension and the Pressure-Overloaded Heart Essential hypertension is a major risk factor of cardiovascular events. Its etiology seems multifactorial and is poorly understood. Here, we did not find a significant effect of TMAO on the development of hypertension in SHRs. Namely, both SHR-Water and SHR-TMAO groups showed an increase in ABP between the 7th and 15th week of life, and there was no difference in SBP, DBP, and MABP in 16-wk-old rats. Hypertension is known to produce a wide range of complications, such as hypertensive angiopathy, kidney failure, and heart failure (HF). Accordingly, in SHRs but not in age-matched WKY rats, we found multiorgan hypertensive angiopathy and water-electrolyte disturbances. HF is a major complication of hypertension (6). Despite significant progress in diagnosis and treatment, the mortality and cost of care attributable to HF remain very high (11). Patients with hypertension-induced heart disease usually present with HF with preserved ejection fraction (diastolic HF) and increased LVEDP, which is associated with cardiomyocyte hypertrophy, cardiac hypertrophy, and cardiac fibrosis (6, 14, 31). In the present study, 60-wk-old SHRs showed characteristic features of hypertension-induced diastolic HF, including significantly increased heart mass, increased LVEDP, increased plasma NT-proBNP and vasopressin levels, mildly reduced stroke volume, and substantial cardiac fibrosis. Interestingly, the hemodynamic and structural changes in the heart started to appear in 16-wk-old SHRs, i.e., 9–10 wk after the onset of the blood pressure surge. Strikingly, SHRs treated with low-dose TMAO for 56 wk showed significantly smaller hemodynamic, biochemical, and histopathological indexes of HF than untreated SHRs. Namely, the SHR-TMAO group had twofold lower LVEDP, significantly lower plasma NT-proBNP, and significantly lower cardiac fibrosis. The positive effect of TMAO was also present in the younger, 16-wk-old SHR-TMAO group. Therefore, our findings strongly suggest that TMAO may have a beneficial effect on the pressure-overloaded heart in hypertensive rats. Several experimental studies have suggested a negative effect of TMAO of the circulatory system (9, 21, 34, 54, 55). However, there are also some studies that have shown potentially beneficial effects of TMAO on the circulatory and the nervous system (5, 7, 20). The discrepancy between our present findings and studies showing a negative effect of TMAO on the heart may result from several factors, such as tested doses of TMAO and experimental settings. For example, Yu et al. (54) showed that TMAO increased the instability of atrial electrophysiology in normal canines; however, in contrast to our study, TMAO was injected locally, i.e., atrial ganglionated plexi of the heart. Savi et al. (34) showed that TMAO exposure worsened cardiomyocyte mechanics and intracellular Ca2+ handling; however, the experiments were performed in vitro. Makrecka-Kuka et al. (21), in in vivo experiments, showed that increased plasma TMAO impairs pyruvate and fatty acid oxidation in cardiac mitochondria in mice. However, in the latter study, mice were treated with several times higher doses of TMAO than in our study, which was associated with a 22- to 23-fold increase in the plasma TMAO level. Finally, Organ et al. (27) found that HF severity was significantly enhanced in transverse aortic constricted mice fed a TMAO-rich diet, which increased plasma TMAO by 16- to 17-fold. It seems that the design of our study may be more suitable for studying the effect of TMAO on the heart function, as data were gathered in the long-term in vivo experiments in which rats were treated with TMAO at a dose that may be achieved in real-life conditions by consuming TMAO-rich food. Specifically, in our study, the concentration of TMAO in the plasma of TMAO-treated rats was only four- to fivefold higher than in untreated rats.

Postulated Mechanism of TMAO Action on the Pressure-Overloaded Heart Research shows that pressure overload of the heart triggers a transition of fibroblast to myofibroblast, producing myocardial fibrosis attributable to accumulation of collagen (14), and that angiotensin and aldosterone independently of hypertension make a major contribution to myocardial fibrosis in hypertensive rats (24). In our study, there were no significant differences between SHR-TMAO and SHR-Water groups in plasma angiotensin II, plasma aldosterone, and cardiac gene expression of angiotensinogen and angiotensin receptors. This suggests a nonangiotensin-dependent mechanism of TMAO effects. We think that the beneficial effect of TMAO on the heart depends on the interaction of TMAO with cardiac proteins, i.e., TMAO acts as a piezolyte. In this regard, a number of biophysical studies have shown that TMAO stabilizes structural proteins, enzymes, DNA, and RNA in conditions of increased hydrostatic pressure, osmotic pressure, temperature, or exposure to denaturants such as urea (5, 8, 10, 19, 26, 28, 29, 39, 46). Furthermore, it is well established that organisms exposed to high hydrostatic and/or osmotic pressures accumulate TMAO to protect their cells from osmotic and hydrostatic pressure stresses (32, 48–50, 52). Certainly, cardiac cells, in particular cardiac cells of subjects with hypertension, are exposed to substantial hydrostatic stress, i.e., significant diastole-systole changes in intraventricular pressure (0–180 mmHg diastole-systole difference or greater). We hypothesized that cardiac cells of rats treated with TMAO are more resilient to hydrostatic stress caused by diastolic-systolic changes in hydrostatic blood pressure, which resulted in preserved biomechanical function of cardiomyocytes and lower fibrosis. Therefore, biophysical studies evaluating the effect of TMAO on pressure-dependent folding of structural proteins and enzymes of cardiomyocytes are needed.

Increased Plasma TMAO and Cardiovascular Risk The issue of a number of clinical studies showing a positive correlation between increased plasma TMAO and an increased cardiovascular risk (18, 30, 35, 36, 42, 44) needs to be addressed. However, some studies have not confirmed such an association (7, 22, 51) or showed that the correlation is dependent on race (37) or point to a high intraindividual variation of plasma TMAO levels over time (17). We found that 60-wk-old hypertensive rats in the SHR-Water group had a significantly higher plasma TMAO level than rats in the normotensive WKY group. However, taken together, our findings suggest that association of an increased plasma TMAO and cardiovascular disturbances may not be a causative relationship. Similarly, increased plasma BNP is not a causative factor of HF but a compensatory response to a failing heart. We hypothesized that in cardiovascular diseases, TMAO is accumulated to protect cells from hydrostatic and osmotic stresses, i.e., pressure overload and water-electrolyte disturbances. Alternatively, increased plasma TMAO may be a marker of other cardiovascular risk factors, such as low glomerular filtration rate (23), dietary habits, e.g., high salt intake (2), or a disturbed gut-blood barrier. Specifically, research suggests that cardiovascular diseases, including HF and hypertension, produce structural and hemodynamic disturbances in the intestines (13, 15, 33), which increases permeability of the gut-blood barrier and facilitates the passage of gut bacterial metabolites, including TMA, a TMAO precursor, to the portal blood (13).

Limitations A limitation of our study is that it was performed on one animal model of hypertension and HF. However, the characteristics of HF in our study were similar to those found in other animal models of hypertension-induced HF, i.e., Dahl salt-sensitive rats (12) and DOCA-salt hypertensive rats (25). Finally, the model of the pressure-overloaded heart that we used in the present study closely resembles a common type of hypertension-induced HF in humans in terms of structural, hemodynamic, and biochemical characteristics (14, 31). This study would be enriched if we had evaluated the effects of chronic, low-dose TMAO treatment in healthy WKY rats. However, earlier, we found that in healthy Sprague-Dawley rats, a 2-wk 100-fold increase in plasma TMAO did not produce significant hemodynamic effects (45).