Heart failure is a common cause of death in industrialized countries with aging populations. Japan, however, has lower rates of heart failure and fewer deaths linked to this disease than the United States or Europe, despite having the highest proportion of elderly people in the world. Dietary differences between these regions may explain the lower rate of heart failure in Japan. The Japanese diet is rich in seafood, which contains nutrients that promote heart health, such as omega-3 fatty acids.

Seafood also contains other compounds, including trimethylamine oxide (TMAO). Fish that live in deep waters undergo high pressures, which can damage their proteins, but TMAO seems to protect the proteins from harm. In humans, eating seafood increases TMAO levels in the blood and urine, but it is unclear what effects this has on heart health. Increased levels of TMAO in the blood are associated with cardiovascular diseases, but scientists are not sure whether TMAO itself harms the heart. A toxic byproduct of gut bacteria called TMA is converted in TMAO in the body, so it is possible that TMA rather than TMAO is to blame.

To assess the effects of dietary TMAO on heart failure, Gawrys-Kopczynska et al. fed the compound to healthy rats and rats with heart failure for one year. TMAO had no effects on the healthy rats. Of the rats with heart failure that were fed TMAO, all of them survived the year, while one third of rats with heart failure that were not fed TMAO died. TMAO-treated rats with heart failure had lower blood pressure and urinated more than untreated rats with the condition.

The experiments suggest that dietary TMAO may mimic the effects of heart failure treatments, which remove excess water and salt and lower pressure on the heart. More studies are needed to confirm whether TMAO has this same effect on humans.

Some clinical studies have shown that increased levels of trimethylamine-oxide (TMAO) in the plasma are associated with an increased risk of adverse cardiovascular events (Tang et al., 2015; Trøseid et al., 2015; Tang et al., 2013). However, other studies have not confirmed this relationship (Meyer et al., 2016; Yin et al., 2015; Stubbs et al., 2019). Furthermore, basic research data regarding the effect of TMAO on the circulatory system are contradictory (Huc et al., 2018; Aldana-Hernández et al., 2019; Collins et al., 2016; Organ et al., 2016; Savi et al., 2018).

In the plasma, TMAO originates from the liver oxidation of trimethylamine (TMA), a product of gut bacteria metabolism of l-carnitine and choline (Zeisel and Warrier, 2017; Ufnal et al., 2015). However, another direct source of TMAO in humans is TMAO-rich seafood (Cheung et al., 2017; Yancey and Siebenaller, 2015). Therefore, populations whose diet are rich in seafood, such as the Japanese, have higher urine TMAO concentrations than those that do not, for example, Americans (Dumas et al., 2006; Holmes et al., 2008). Interestingly, prevalence and mortality rates of heart failure (HF) are lower in Japan compared to the US or Europe, despite the fact that Japan has the highest proportion of elderly people in the world (Nagai et al., 2018; Ogawa et al., 2007).

Marine animals living in deep water, and thus exposed to hydrostatic pressure stress (HPS), accumulate TMAO (Yancey and Siebenaller, 2015; Ma et al., 2014; Yancey and Siebenaller, 1999; Sarma and Paul, 2013; Yin et al., 2017). Data from biophysical experiments suggest a protective effect of TMAO on cell proteins exposed to HPS. For example, TMAO has been found to stabilize teleost and mammalian lactate dehydrogenase (LDH), a complex tetramer protein that plays an essential role in cellular metabolism (Yancey and Siebenaller, 1999).

Notably, in a heart exposed to catecholamine-induced stress or in a hypertensive heart, diastolic-systolic changes in pressure may exceed 220 mmHg. These fluctuations in pressure can happen in humans up to 200 times per minute. These numbers are even higher in small animals. These events may produce an environment in which the hydrostatic pressure changes approximately 100 000 times in 24 hr. However, the effect of HPS produced by the contracting heart on cardiac proteins is obscure.

Recently, we hypothesized that TMAO may benefit the circulatory system by protecting cardiac LDH exposed to HPS produced by diastole-systole-driven changes in the hydrostatic pressure of the contracting heart (Ufnal and Nowiński, 2019). Here, we investigated whether a continuous, 12-months-long oral administration of TMAO in healthy Sprague-Dawley rats (SD), in SD exposed to catecholamine stress, and in animal model of heart failure (HF) with reduced ejection fraction (SHHF) exerts beneficial effects on the circulatory system. Furthermore, we evaluated whether TMAO protects the protein structure of cardiac LDH exposed to HPS. To examine the effects of HPS in the context that mimics the environment produced by the contracting heart, we developed a novel experimental system using microfluidics chambers with piezoelectric valves and pressure controllers.

Spontaneously Hypertensive Heart Failure (SHHF) and age-matched Sprague-Dawley (SD) rats were randomly assigned to either the control group (rats drinking tap water) or the TMAO group (rats drinking TMAO solution in tap water). At the age of 56 weeks, the ISO-control and ISO-TMAO series were given isoprenaline at a dose of 100 mg/kg b.w. to produce catecholamine stress. The experimental protocol is depicted in Figure 1.

Figure 1

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SD rats

In general SD rats showed no pathological findings (Table 1, Figures 2 and 3, and Figure 3—figure supplement 1).

Figure 2

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Table 1

Group/Parameter		SD-control	SD-TMAO	P
Survival, energy and water balance
Survival from the study onset (%, n)		100% (10/10)	100% (10/10)	-
Body mass (g)		446.4 ± 40.63	452.86 ± 37.11	0.36
24 hr food intake (g)		19.99 ± 2.83	21.49 ± 2.64	0.12
24 hr water intake (g)		31.56 ± 10.02	36.66 ± 5.18	0.09
24 hr urine output (g)		18.66 ± 2.35	22.66 ± 6.49	0.04
Tibia length (cm)		4.31 ± 0.1	4.25 ± 0.15	0.15
TMAO
Plasma TMAO (µmol/L)		6.55 ± 0.65	39.73 ± 20.6	<0.001
24 hr TMAO urine excretion (µmoles)		5.96 ± 1.49	103.05 ± 56.7	<0.001
Heart mass
Heart mass (g)		1.44 ± 0.08	1.46 ± 0.14	0.38
Arterial blood pressure and heart rate
Systolic (mmHg)		129.72 ± 8.56	127.07 ± 5.84	0.87
Diastolic (mmHg)		80.56 ± 13.3	86.61 ± 9.74	0.15
HR (beats/min)		333.9 ± 45	364.4 ± 8.6	0.04
Echocardiographic parameters
LVEDV (mL)		0.47 ± 0.15	0.57 ± 0.1	0.06
LVESV (mL)		0.12 ± 0.04	0.13 ± 0.03	0.44
IVSs (cm)		0.35 ± 0.03	0.35 ± 0.03	0.42
IVSd (cm)		0.24 ± 0.03	0.25 ± 0.03	0.19
SV (mL)		0.36 ± 0.11	0.44 ± 0.1	0.04
EF (%)		75.63 ± 3.02	77.13 ± 6.01	0.27
Left ventricle hemodynamic parameters (direct measurements)
LVEDP (mmHg)		4.12 ± 0.78	4.25 ± 0.92	0.45
dP/dt (mmHg/ms)		6.54 ± 1.02	7.31 ± 1.23	0.12
-dP/dt (mmHg/ms)		5.00 ± 0.69	5.26 ± 0.45	0.21
Plasma NT-proBNP
NT-proBNP (pg/mL)		24.79 ± 8.1	18.61 ± 8.17	0.22
Electrolyte balance
Serum sodium (mmol/L)		138.86 ± 2.27	138.44 ± 0.50	1.0
24 hr sodium urine excretion (mmoles)		1.76 ± 0.23	2.11 ± 0.08	0.003
Serum potassium (mmol/L)		5.27 ± 0.89	5.13 ± 0.17	0.71
24 hr potassium urine excretion (mmoles)		2.83 ± 0.58	3.00 ± 0.12	0.23
Serum creatinine clearance (mL/min)		1.15 ± 0.18	1.16 ± 0.07	0.67
Hormones
Angiotensin II (pg/mL)		244.93 ± 35.55	250.07 ± 64.95	0.43
Aldosterone (pg/mL)		897.05 ± 95.34	925.61 ± 75.29	0.27
Vasopressin (ng/mL)		0.92 ± 0.98	1.78 ± 0.59	0.02

Table 1—source data 1

Metabolic, renal and cardiovascular parameters in 58-week-old normotensiveSD rats.

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SD rats: Control vs TMAO treatment

Survival and water-electrolyte balance

There was no significant difference between the SD-control and SD-TMAO rats in survival rate (100% in both groups), body mass and food intake. The SD-TMAO rats showed 5-times higher plasma TMAO levels than the SD-control group. The SD-TMAO group showed a significantly higher 24 hr urine output and sodium urine excretion. There was no significant difference between the SD-TMAO and SD-control group in plasma Ang II and aldosterone level. However, the SD-TMAO rats showed higher vasopressin concentration (Table 1).

Circulatory parameters

There was no significant difference between the SD-control and SD-TMAO group in hemodynamic parameters measured directly and with echocardiography, however, the SD-TMAO rats showed higher SV and HR (Table 1).

Histopathology

There were no pathological changes in the heart, lungs and kidneys in either the SD-control or SD-TMAO group (Figure 3 and Figure 3—figure supplement 1).

Gene expression

The SD-TMAO rats showed significantly lower expression of AT1 receptors (AT1R) in the heart. In the kidneys, SD-TMAO rats showed significantly higher expression of renin but lower expression of AT2 receptors (AT2R) and a trend towards a lower expression of AT1R (Figures 4 and 5).

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Real-time RT-PCR analysis, expression profiles in heart.

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Real-time RT-PCR analysis, expression profiles in kidneys.

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SHHF rats

In general, the SHHF animals showed characteristics of hypertrophic cardiomyopathy with compromised systolic function including substantially increased heart mass and plasma NT-proBNP, decreased stroke volume and ejection fraction as well as lung edema (Table 2, Figure 2). Histological evaluation of the heart revealed dilated cardiomyopathy that is a moderate increase in the diameter of cardiomyocytes, enlargement of the nucleus and a reduction of cytoplasmic acidity (Figure 6). In the lungs, a passive hyperemia with thickening of the interalveolar septa, a weak focal parenchymal edema and a moderate stromal connective tissue hyperplasia were present. There were no significant pathological changes in the kidneys (Figure 6).

Table 2

Group/ Parameter		SHHF-control	SHHF-TMAO	P
Survival, Energy and water balance
Survival from the onset of the study (%, n)		66% (6/9)	100% (9/9)	0.07 #
Body mass (g)		475.2 ± 17.1	476.3 ± 12.1	0.43
24 hr food intake (g)		23.2 ± 3.2	24.2 ± 2.3	0.26
24 hr water intake (mL)		37.5 ± 7.5	41.1 ± 6.6	0.17
24 hr urine output (mL)		14.8 ± 2.8	17.9 ± 2.5	0.02
Tibia length (cm)		3.95 ± 0.21	3.99 ± 0.11	0.34
TMAO
Plasma TMAO (µmol/L)		6.71 ± 1.49	20.32 ± 7.21	<0.001
24 hr TMAO urine excretion (µmoles)		9.97 ± 3.46	126.8 ± 32.8	<0.001
Heart mass
Heart mass (g)		1.87 ± 0.31	1.72 ± 0.3	0.19
Arterial blood pressure and heart rate
Systolic (mmHg)		136.2 ± 12.8	126.8 ± 12.7	0.11
Diastolic (mmHg)		98.6 ± 7.3	87.6 ± 5.6	0.004
HR (beats/min)		314 ± 61	302 ± 20	0.31
Echocardiographic parameters
LVEDV (mL)		0.37 ± 0.19	0.52 ± 0.20	0.11
LVESV (mL)		0.14 ± 0.08	0.15 ± 0.1	0.41
IVSs (cm)		0.41 ± 0.01	0.35 ± 0.09	0.21
IVSd (cm)		0.29 ± 0.05	0.27 ± 0.07	0.28
SV (mL)		0.24 ± 0.12	0.36 ± 0.12	0.053
EF (%)		64 ± 8.5	71 ± 6.1	0.06
Left ventricle hemodynamic parameters (direct measurements)
LVEDP (mmHg)		3.10 ± 0.78	3.41 ± 1.95	0.87
dP/dt (mmHg/ms)		5.88 ± 0.92	5.50 ± 0.98	0.41
-dP/dt (mmHg/ms)		2.35 ± 0.28	2.55 ± 0.67	0.27
Plasma NT-proBNP
NT-proBNP (pg/mL)		52.26 ± 0.15.0	42.80 ± 9.5	0.09
Electrolyte balance
Serum sodium (mmol/L)		142.4 ± 3.31	138.9 ± 2.98	0.04
24 hr sodium urine excretion (mmoles)		1.42 ± 0.28	1.93 ± 0.33	0.005
Serum potassium (mmol/L)		4.73 ± 0.33	4.49 ± 0.28	0.09
24 hr potassium urine excretion (mmoles)		2.89 ± 0.42	3.40 ± 0.54	0.04
Serum creatinine clearance (mL/min)		0.42 ± 0.17	0.53 ± 0.05	0.06
Hormones
Angiotensin II (pg/mL)		325.7 ± 39.8	276.7 ± 38.3	0.02
Aldosterone (pg/mL)		816.8 ± 300.4	758.4 ± 142.8	0.32
Vasopressin (ng/mL)		3.02 ± 1.24	3.11 ± 1.03	0.45
Cytokines
TNF-a (pg/mL)		34.56 ± 24.69	24.98 ± 7.92	0.19
IL-10 (pg/mL)		15.91 ± 4.66	28.17 ± 14.39	0.036

Table 2—source data 1

Metabolic, renal and cardiovascular parameters in SHHF rats.

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Percentage of myocardial fibrosis in SHHF rats.

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SHHF rats: Control vs TMAO treatment

Survival and water-electrolyte balance

All SHHF-TMAO rats (n = 9) survived from the beginning of the experiment till the age of 58 weeks, that is the time of anesthesia before echocardiography. In contrast, three out of nine animals (33%) in the SHHF-control group died. Specifically, two rats were euthanized due to hemiparalysis (ischemic stroke) and dyspnea (post-mortem lung edema), and one died spontaneously (post-mortem lung edema), at the age of 52, 56 and 57 weeks, respectively. The log-rank test showed a trend (p=0.0651) towards higher survival in the SHHF-TMAO than in SHHF-control group (Figure 7). One SHHF-TMAO animal died during anesthesia before echocardiographic examination. The following numbers in each group were included for further analysis: SHHF-TMAO (n = 8) and SHHF-control (n = 6).

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There was no significant difference between the groups in food intake. The SHHF-TMAO animals showed 3–4-fold higher plasma TMAO level than the SHHF-control rats. The SHHF-TMAO rats had a significantly higher 24 hr urine output and sodium excretion than the SHHF-control rats. The SHHF-TMAO group showed significantly lower plasma sodium and Ang II levels, but there were no differences in aldosterone and vasopressin plasma levels (Table 2).

Circulatory parameters

The SHHF-control and SHHF-TMAO group showed hypertrophic cardiomyopathy with compromised systolic functions and increased plasma NT-proBNP (Figures 2 and 6, Table 2). The SHHF-TMAO rats had a significantly lower diastolic blood pressure and a trend towards lower plasma NT-proBNP (p=0.09), higher stroke volume (p=0.053) and higher ejection fraction (p=0.06), (Table 2).

Histopathology

The general histological picture of myocardium and cardiomyocyte morphology did not differ significantly between the groups. Morphometric analysis did not show significant differences in the degree of myocardial fibrosis between the groups. However, in the TMAO group there was a trend towards less myocardial fibrosis (p=0.07), cardiomyocyte bands were more visible, and smaller inflammatory infiltration was found. The histological picture of the lungs and kidneys did not differ significantly between SHHF-control and SHHF-TMAO groups (Figure 6).

Gene expression

The SHHF-TMAO rats had a significantly lower expression of AT1R and a significantly higher expression of AT2R in the heart. In the kidneys, the SHHF-TMAO rats showed a significantly higher expression of renin and lower expression of AT1AR (Figures 4 and 5).

SD-ISO rats

In general, the SD animals subjected to catecholamine stress (isoprenaline) showed some characteristics of Takotsubo-like cardiomyopathy, including a mild degree of apical akinesis/dyskinesis, edema of cardiomyocytes, increased NT-proBNP level and mild lung edema (Figures 2 and 8, Table 3). Numerous, scattered foci of banded mononuclear cell infiltration were present in the myocardium. Severe hyperemia of myocardial capillaries and arterioles and small organized foci of myocardial extravasation were present. Nevertheless, the majority of the myocardium remained normal in structure. The lungs presented transudate in the alveoli. In the kidneys, there was a weak congestion in the medulla and renal bodies. A small number of tubules filled with an acidic substance was present. Stimulation of stromal fibrocytes without production of connective tissue fibers was observed (Figure 8).

Table 3

Group/ Parameter		ISO-control	ISO-TMAO	P
Survival, Energy and water balance
Survival from the study onset (%, n)		90% (9/10)	100% (10/10)	0.32#
Body mass (g)	T1	434.44 ± 22.93	432.69 ± 37.59	0.45
	T3	438.67 ± 23.99	428.71 ± 37.14	0.25
24 hr food intake (g)	T1	21.54 ± 1.43	21.84 ± 1.94	0.51
	T3	21.24 ± 2.44	22.08 ± 1.91	0.21
24 hr water intake (mL)	T1	34.99 ± 3.10	34.82 ± 3.89	0.53
	T3	34.77 ± 5.47	35.59 ± 1.92	0.34
24 hr urine output (g)	T1	19.28 ± 2.73	20.03 ± 4.42	0.34
	T3	19.68 ± 4.29	20.23 ± 2.6	0.37
Tibia length (cm)	T3	4.33 ± 0.06	4.34 ± 0.1	0.35
TMAO
Plasma TMAO (µmol/L)	T3	5.95 ± 2.35	32.51 ± 11.43	<0.001
24 hr TMAO urine excretion (µmoles)	T3	5.74 ± 1.64	119.12 ± 65.96	<0.001
Heart mass
Heart mass (g)	T3	1.46 ± 0.13	1.43 ± 0.19	0.39
Arterial blood pressure and heart rate
Systolic (mmHg)	T3	142.48 ± 10.63	130.92 ± 11.67	0.026
Diastolic (mmHg)	T3	97.10 ± 9.95	87.59 ± 10.47	0.037
HR (beats/min)	T3	356.56 ± 23.39	344.19 ± 49.69	0.26
Echocardiographic parameters
LVEDV (mL)	T1	0.44 ± 0.22	0.33 ± 0.17	0.15
	T2	0.54 ± 0.22	0.53 ± 0.23	0.47
	T3	0.51 ± 0.11	0.53 ± 0.34	0.21
LVESV (mL)	T1	0.13 ± 0.09	0.11 ± 0.07	0.31
	T2	0.22 ± 0.13	0.13 ± 0.05	0.054
	T3	0.12 ± 0.05	0.08 ± 0.02	0.01
IVSs (cm)	T1	0.33 ± 0.03	0.32 ± 0.05	0.29
	T2	0.33 ± 0.06	0.35 ± 0.09	0.29
	T3	0.36 ± 0.04	0.32 ± 0.05	0.07
IVSd (cm)	T1	0.24 ± 0.04	0.23 ± 0.04	0.28
	T2	0.26 ± 0.03	0.25 ± 0.04	0.41
	T3	0.24 ± 0.04	0.25 ± 0.03	0.31
SV (mL)	T1	0.29 ± 0.15	0.26 ± 0.12	0.40
	T2	0.35 ± 0.17	0.47 ± 0.22	0.09
	T3	0.38 ± 0.09	0.34 ± 0.07	0.69
EF (%)	T1	71.22 ± 6.12	72.11 ± 14.51	0.45
	T2	73.11 ± 8.88	70.11 ± 12.84	0.28
	T3	78.67 ± 7.18	77.89 ± 11.94	0.43
Left ventricle hemodynamic parameters (direct measurements)
LVEDP (mmHg)	T3	6.73 ± 2.55	4.46 ± 0.74	0.03
dP/dt (mmHg/ms)	T3	9.20 ± 1.54	6.55 ± 1.18	0.004
-dP/dt (mmHg/ms)	T3	5.19 ± 0.38	4.76 ± 0.65	0.09
Plasma NT-proBNP
NT-proBNP (pg/mL)	T3	64.49 ± 43.59	22.01 ± 22.83	0.02
Electrolyte balance
Serum sodium (mmol/L)	T3	136.88 ± 3.56	137.89 ± 1.62	0.23
24 hr sodium urine excretion (mmoles)	T3	1.93 ± 0.27	2.09 ± 0.42	0.18
Serum potassium (mmol/L)	T3	5.53 ± 0.89	5.02 ± 0.77	0.11
24 hr potassium urine excretion (mmoles)	T3	2.56 ± 0.35	2.92 ± 0.35	0.03
Serum creatinine clearance (mL/min)	T3	1.26 ± 0.23	1.24 ± 0.26	0.43
Hormones
Angiotensin II (pg/mL)	T3	286.4 ± 24.4	272.6 ± 39.5	0.35
Aldosterone (pg/mL)	T3	938.6 ± 114.6	1032.4 ± 120.6	0.07
Vasopressin (ng/mL)	T3	0.98 ± 0.55	1.28 ± 0.66	0.18

Table 3—source data 1

Metabolic, renal and cardiovascular parameters in SD-ISO rats.

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Percentage of necrotic and inflammatory area in myocardium of SD-ISO rats.

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SD-ISO rats: Control vs TMAO treatment

Survival and water-electrolyte balance

There was no significant difference between ISO-control and ISO-TMAO groups in survival rate (9/10 vs 10/10, respectively). The ISO-TMAO rats showed five times higher TMAO plasma level than the ISO-control rats. There was no significant difference in food and water intake between the groups. There was also no significant difference between the groups in 24 hr urine output, however, the ISO-TMAO rats tended to have higher natriuresis (Table 3).

Circulatory parameters

The ISO-TMAO rats showed significantly lower systolic and diastolic blood pressure, significantly lower plasma NT-proBNP and lower LV ESV and LVEDP (Table 3).

Histopathology

The histological picture of the heart, lungs and kidneys did not differ significantly between the groups (Figure 8).

Gene expression

In the heart, the ISO-TMAO rats showed significantly lower expression of ATG, AT1R and AT2R. In the kidneys, the ISO-TMAO rats showed significantly lower expression of ATG and AT1R and significantly higher expression of renin (Figures 4 and 5).

Effect of TMAO on renal excretion in SD rats - acute studies

We evaluated changes in renal excretion induced by TMAO, urea and saline intravenous administration in acute experiments. Results are summarized in Figure 9. Only TMAO induced diuresis. The pattern of diuresis and total solutes excretion induced by TMAO were similar. Increases of V and UosmV induced by TMAO were associated with transient decrease of Uosm, whereas UNaV and UKV were not affected. This indicates that TMAO did not affected the tubular transport of sodium and potassium but induced osmotic diuresis. The bolus infusions of TMAO or saline produced a transient increase in ABP with no changes in HR, which was followed by a decrease in ABP below the baseline (by 6 ± 3 mmHg). There was no significant correlation between changes in ABP and diuresis.

Figure 9

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Effects of TMAO, urea and saline on renal excretion.

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Effect of TMAO on structure of LDH exposed to HPS and increased temperature

Labeled LDH was stable in PBS solution, showing no tendency for spontaneous aggregation. The value of diffusion coefficients measured by FCS at 25°C was 49.2 ± 3.3 μm2/s. This corresponds to a hydrodynamic radius of around 5.0 nm, which is in line with previously reported values (Zipper and Durchschlag, 1998).

The tertiary and quaternary structures of LDH, with and without TMAO, were not influenced by a 24 hr treatment with HPS (pressure oscillations mimicking those of a rat heart), (Figure 10A). Tests performed using a different pressure oscillation system, where pressures up to 1000 mmHg were applied, did not detect observable changes in the protein structure (see Figure 10—figure supplement 1).

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Figure 10—source data 1

LDH incubated at a constant atmospheric pressure for 24 hrs or LDH exposed to oscillating pressure for 24 hrs with or without TMAO.

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The incubation of LDH at atmospheric pressure and elevated temperatures (50–80°C) changed the diffusion coefficient of LDH, indicating the dissociation of LDH tetramers, as well as protein denaturation and aggregation. The addition of 1M TMAO produced a moderate stabilizing effect on LDH, shifting the threshold of observed protein morphology change towards higher temperatures (Figure 10B). Specifically, it seems that the gradual dissociation of tetramers to monomers occurred above 55°C, which was followed by the denaturation of the tertiary protein structure at higher temperatures. At a concentration of LDH >3 nM, the aggregation of monomers prevailed. At a lower concentration, the aggregation progressed more slowly, if at all, while any aggregates that did form, were too sparse to influence the measurement results. Further work, including probing a broader matrix of LDH concentrations and temperatures, is needed to confirm these initial findings. Nevertheless, in all the experiments, presence of TMAO shifted the threshold of change in LDH morphology towards higher temperatures and diminished the magnitude of the change. This suggests a stabilizing effect of TMAO on the native structure of the protein.

Our study provides evidence that TMAO exerts a beneficial effect in heart failure rats. The beneficial effect of TMAO is associated with diuretic, natriuretic and hypotensive actions rather than a stabilizing effect on cardiac LDH. Specifically, we found that HPS at the magnitude substantially greater than that generated by a contracting heart did not affect cardiac LDH protein structure with or without TMAO.

Plasma TMAO originates from trimethylamine (TMA), a product of gut bacteria, which is oxidized by the liver to form TMAO. It can also be obtained from dietary TMAO-rich seafood (Zeisel and Warrier, 2017; Ufnal et al., 2015). Several years ago, Tang and collaborators showed that increased plasma TMAO is associated with an increased risk of major adverse cardiovascular events (Tang et al., 2013), which generated numerous clinical and experimental studies, suggesting negative effects of TMAO (for review, [Zeisel and Warrier, 2017; Onyszkiewicz et al., 2020]). However, other clinical studies did not find an association between high plasma TMAO and increased cardiovascular risk (Meyer et al., 2016; Yin et al., 2015; Stubbs et al., 2019).

Our previous studies suggest that TMAO is only a surrogate marker and that the toxic cardiovascular effects are caused not by TMAO itself, but by a TMAO precursor, that is TMA (Jaworska et al., 2019a; Jaworska et al., 2019b; Jaworska et al., 2019c). Notably, the latter is a well-established toxin (Pospischil et al., 2017). Since TMA is oxidized (likely, detoxified) by the liver to TMAO, the increased plasma TMAO is simply a proxy to the plasma levels of the toxic TMA. What is more, recent experimental studies provide evidence for a beneficial action of TMAO at low doses (Huc et al., 2018; Aldana-Hernández et al., 2019; Collins et al., 2016).

Here, we investigated the effect of TMAO in (i) healthy SD rats, (ii) SHHF (SHHF/MccGmiCrl-Lepr^cp/Crl) which showed characteristics of heart failure with compromised systolic function and (iii) SD subjected to catecholamine stress (isoprenaline).

Our findings show that increased dietary TMAO, which elevates plasma TMAO level 3–5-fold, increased diuresis and natriuresis in SD and SHHF rats. This is associated with reduced mortality and favorable hemodynamic and biochemical changes in HF rats.

In this study, during a stress-free 52 week observation, the fatality rate of the SHHF animals treated with TMAO was 0% in comparison to 33% in the untreated group. This was associated with several beneficial hemodynamic and biochemical characteristics in the TMAO-treated SHHF rats, namely; a lower diastolic BP, higher diuresis and natriuresis, a lower expression of AT1R in the heart with concomitant increase in AT2R, and a trend towards lower cardiac fibrosis. Regarding the latter, the tissue angiotensin system contributes significantly to remodeling of the heart, and its inhibition is critical in the treatment of heart failure (Leong et al., 2019).

Some of the characteristic features of HF are inflammation and increased sympathetic activity. In this regard, the SHHF-TMAO group had a significantly increased plasma level of IL-10, an anti-inflammatory cytokine, and a trend towards lower plasma TNF-α, a pro-inflammatory mediator. Significantly lower diastolic blood pressure together with mildly lower heart rate may also suggest lower sympathetic activity in the SHHF-TMAO group. However, to fully address this issue further studies evaluating concentration of catecholamines metabolites in urine are needed. It is worth stressing that due to the high mortality in the SHHF-control group, the final analysis of hemodynamic and biochemical parameters included only the survivors that is the healthiest rats. It is interesting to speculate, had all the SHHF-control rats survived to the experimental endpoints, whether the differences between the SHHF-TMAO and SHHF-control would have been even greater.

Finally, a key characteristic of HF is fluid retention. We think that the beneficial effects of TMAO described above were secondary to the TMAO-produced increase in diuresis and natriuresis, which is a cornerstones of HF treatment.

We assumed that the diuretic and natriuretic effects of TMAO resulted from osmotic diuresis. To evaluate the notion, we performed additional acute experiments in rats. We found that intravenous administration of TMAO, but not saline given at the same volume, significantly increased diuresis in anesthetized rats. Furthermore, in chronic studies, TMAO-treated rats showed increased expression of renin in the kidneys. This is a characteristic response to osmotic diuresis, associated with a decreased concentration of sodium in filtrate reaching distal convoluted tubule. Increased diuresis in TMAO-treated rats was present despite elevated plasma levels of vasopressin which allows the reabsorption of water from the filtrate in collecting ducts. The reabsorption is driven by the osmotic pressure gradient between the filtrate and the kidney medulla. In TMAO-treated rats, the osmotic gradient was likely decreased due to the high osmotic activity of TMAO and high TMAO levels in the filtrate. This could decrease the reabsorption of water from the filtrate despite the vasopressin-induced increase in the permeability of collecting ducts to water.

To evaluate diuretic effect of TMAO, we compared TMAO to urea, which was used as a diuretic treatment of advanced HF before being replaced by thiazide diuretics (Shanoff, 1970; Crawford, 1925). Notably, both molecules are nitrogenous compounds, have a similar molecular weight, and play the role of an osmolyte in animals (Yancey and Siebenaller, 2015). In our experiments, TMAO produced significant diuresis whereas urea did not. This was likely due the fact that we evaluated the same (equimolar) doses of urea and TMAO, whereas physiological concentrations of TMAO in the plasma is radically lower (micromoles/L) in comparison to urea (millimoles/L). Therefore, our findings suggest that TMAO exerts a significantly more potent diuretic effect than urea.

A number of biological and biophysical studies indicate that TMAO is not only an osmolyte but also a piezolyte, that is stabilizes structural proteins and enzymes, such as LDH, in conditions of increased hydrostatic pressure (Yancey and Siebenaller, 1999; Sarma and Paul, 2013; Yin et al., 2017). Therefore, the accumulation of TMAO in the bodies of deep-water animals may protect cell proteins from HPS (Yancey and Siebenaller, 2015; Yancey and Siebenaller, 1999).

The effect of HPS produced by the contracting heart on cardiac proteins is unclear. Based on studies showing that TMAO stabilizes teleost and mammalian lactate dehydrogenases against inactivation by hydrostatic pressure (Yancey and Siebenaller, 1999; Al-Ayoubi et al., 2019; Yancey et al., 2001), we hypothesized that TMAO may exert a protective effect on the circulatory system by stabilizing the protein structure of cardiac LDH, a complex enzyme that plays essential role in cardiac metabolism (Ufnal and Nowiński, 2019).

To evaluate this hypothesis, we built a unique experimental setup to mimic the changes in hydrostatic pressure that occur in the heart under conditions of catecholamine stress. These in vitro experiments showed that changes in hydrostatic pressure generated by the contracting heart, and even much higher pressures, do not disturb the protein structure of cardiac LDH. Finally, the addition of TMAO did not produce any effect, suggesting neither positive nor negative effect of TMAO on LDH exposed to HPS. Nevertheless, we showed that TMAO stabilized LDH exposed to other denaturant, that is high temperature, which is in line with other studies (Schummel et al., 2018). In general, changes in protein structure lead to perturbed (or even the loss of) activity of proteins. However, further studies are needed to evaluate if LDH activity would be affected by HPS, TMAO and temperature in a similar manner as LDH protein structure.

Altogether, our study shows that TMAO exerts beneficial effects in cardiovascular pathologies associated with fluid retention such as HF. The beneficial effects of TMAO appear to stem from its diuretic action rather than from the protein-stabilizing effect of TMAO on LDH, which was described for deep-sea animals, but was found not be involved here. It may be that the hydrostatic pressure generated by a contracting heart is far lower than the deep‐sea pressures and is kinetic (pulsing and moving) rather than static. In this regard, there is evidence that static pressure may have very different effects on cells and proteins than kinetic pressure (Kaarniranta et al., 1998). Nevertheless, the stabilizing effect of TMAO on other, less stable, cardiac proteins exposed to HPS cannot be excluded.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures and tables.

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Author details

1. Marta Gawrys-Kopczynska

   Department of Experimental Physiology and Pathophysiology, Laboratory of the Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland

   Contribution

   Data curation, Formal analysis, Writing - original draft

   Competing interests

   No competing interests declared

2. Marek Konop

   Department of Experimental Physiology and Pathophysiology, Laboratory of the Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland

   Contribution

   Data curation, Formal analysis, Writing - original draft

   Competing interests

   No competing interests declared

3. Klaudia Maksymiuk

   Department of Experimental Physiology and Pathophysiology, Laboratory of the Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland

   Contribution

   Data curation, Formal analysis, Writing - original draft

   Competing interests

   No competing interests declared

4. Katarzyna Kraszewska

   Department of Experimental Physiology and Pathophysiology, Laboratory of the Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland

   Contribution

   Data curation, Formal analysis, Writing - original draft

   Competing interests

   No competing interests declared

5. Ladislav Derzsi

   Department of Soft Condensed Matter, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland

   Contribution

   Conceptualization, Data curation, Formal analysis, Writing - original draft

   Competing interests

   No competing interests declared

6. Krzysztof Sozanski

   Department of Soft Condensed Matter, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland

   Contribution

   Conceptualization, Data curation, Formal analysis, Writing - original draft

   Competing interests

   No competing interests declared

7. Robert Holyst

   Department of Soft Condensed Matter, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland

   Contribution

   Conceptualization, Data curation, Formal analysis, Writing - original draft

   Competing interests

   No competing interests declared

8. Marta Pilz

   Department of Soft Condensed Matter, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland

   Contribution

   Data curation, Formal analysis, Writing - original draft

   Competing interests

   No competing interests declared

9. Emilia Samborowska

   Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

10. Leszek Dobrowolski

    Department of Renal and Body Fluid Physiology, M. Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

    Contribution

    Data curation, Formal analysis, Writing - original draft

    Competing interests

    No competing interests declared

11. Kinga Jaworska

    Department of Experimental Physiology and Pathophysiology, Laboratory of the Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland

    Contribution

    Data curation, Formal analysis, Writing - original draft, Writing - review and editing

    Competing interests

    No competing interests declared

12. Izabella Mogilnicka

    Department of Experimental Physiology and Pathophysiology, Laboratory of the Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland

    Contribution

    Data curation, Formal analysis

    Competing interests

    No competing interests declared

13. Marcin Ufnal

    Department of Experimental Physiology and Pathophysiology, Laboratory of the Centre for Preclinical Research, Medical University of Warsaw, Warsaw, Poland

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    mufnal@wum.edu.pl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0088-8284

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