Trimethylamine- N-oxide acutely increases cardiac muscle contractility

Abstract

Cardiovascular disease is a major cause of morbidity and mortality among patients with chronic kidney disease (CKD). Trimethylamine-N-oxide (TMAO), a uremic metabolite that is elevated in the setting of CKD, has been implicated as a nontraditional risk factor for cardiovascular disease. While association studies have linked elevated plasma levels of TMAO to adverse cardiovascular outcomes, its direct effect on cardiac and smooth muscle function remains to be fully elucidated. We hypothesized that pathological concentrations of TMAO would acutely increase cardiac and smooth muscle contractility. These effects may ultimately contribute to cardiac dysfunction during CKD. High levels of TMAO significantly increased paced, ex vivo human cardiac muscle biopsy contractility (P < 0.05). Similarly, TMAO augmented contractility in isolated mouse hearts (P < 0.05). Reverse perfusion of TMAO through the coronary arteries via a Langendorff apparatus also enhanced cardiac contractility (P < 0.05). In contrast, the precursor molecule, trimethylamine (TMA), did not alter contractility (P > 0.05). Multiphoton microscopy, used to capture changes in intracellular calcium in paced, adult mouse hearts ex vivo, showed that TMAO significantly increased intracellular calcium fluorescence (P < 0.05). Interestingly, acute administration of TMAO did not have a statistically significant influence on isolated aortic ring contractility (P > 0.05). We conclude that TMAO directly increases the force of cardiac contractility, which corresponds with TMAO-induced increases in intracellular calcium but does not acutely affect vascular smooth muscle or endothelial function of the aorta. It remains to be determined if this acute inotropic action on cardiac muscle is ultimately beneficial or harmful in the setting of CKD.NEW & NOTEWORTHY We demonstrate for the first time that elevated concentrations of TMAO acutely augment myocardial contractile force ex vivo in both murine and human cardiac tissue. To gain mechanistic insight into the processes that led to this potentiation in cardiac contraction, we used two-photon microscopy to evaluate intracellular calcium in ex vivo whole hearts loaded with the calcium indicator dye Fluo-4. Acute treatment with TMAO resulted in increased Fluo-4 fluorescence, indicating that augmented cytosolic calcium plays a role in the effects of TMAO on force production. Lastly, TMAO did not show an effect on aortic smooth muscle contraction or relaxation properties. Our results demonstrate novel, acute, and direct actions of TMAO on cardiac function and help lay the groundwork for future translational studies investigating the complex multiorgan interplay involved in cardiovascular pathogenesis during CKD.

Keywords: calcium homeostasis; cardiac muscle; cardiovascular disease; chronic kidney disease; vascular smooth muscle.

Fig. 1.

Trimethylamine-N-oxide (TMAO) enhances human cardiac muscle contractility. A: raw tracings of paced, human cardiac biopsy contractions at baseline and following TMAO. B: change in isometric tension normalized to baseline contractions (n = 4 to 5). C: change in maximum slope normalized to baseline contractions (n = 4 to 5). D: change in minimum slope normalized to baseline contractions (n = 4 to 5). *P < 0.05, statistical difference from vehicle. TMAO; 3 mM.

Fig. 2.

Bath application of trimethylamine-N-oxide (TMAO) increases cardiac contractility in isolated mouse hearts. A: raw tracings of paced, ventricular contractions at baseline and following 3 mM TMAO. B: change in isometric tension normalized to baseline contractions (n = 6 to 7). C: change in maximum slope normalized to baseline contractions (n = 6 to 7). D: change in minimum slope normalized to baseline contractions (n = 6 to 7). *P < 0.05, statistical difference from vehicle. †P < 0.05, statistical difference from 300 µM TMAO.

Fig. 3.

Perfusion of the coronary circulation with trimethylamine-N-oxide (TMAO) increases contractility. A: Langendorff perfusion apparatus: a cannula inserted into the aorta facilitates perfusion of the coronary circulation while the heart is connected to the force transducer and hung between bipolar stimulating electrodes. B: change in isometric tension normalized to baseline contractions (n = 4–13). C: change in maximum slope normalized to baseline contractions (n = 4–13). D: change in minimum slope normalized to baseline contractions (n = 4–13). E: change in isometric tension normalized to baseline contraction after perfusion with d-mannitol. *P < 0.05, statistical difference from vehicle.

Fig. 4.

Perfusion of the coronary circulation with trimethylamine (TMA) does not alter contractility. A: change in isometric tension normalized to baseline contractions (n = 4 to 5). B: change in maximum slope normalized to baseline contractions (n = 4 to 5). C: change in minimum slope normalized to baseline contractions (n = 4 to 5).

Fig. 5.

Trimethylamine-N-oxide (TMAO) increases intracellular calcium in isolated mouse hearts. A: whole hearts were paced and cellular Fluo-4 AM fluorescence measured in the left ventricle using two-photon microscopy following treatments with TMAO or vehicle. The color scale represents increasing fluorescence values from 0 to 255 (maximum). B: change in the average Fluo-4 AM fluorescence, normalized to baseline fluorescence (n = 4 to 5). *P < 0.05, statistical difference from vehicle.

Fig. 6.

Trimethylamine-N-oxide (TMAO) has no effect on isolated aortic ring contractility. A: representative aortic myography record indicating contractile tension during 30 min perfusion with vehicle or TMAO, followed by 1 µM phenylephrine (PE) to induce contraction, and 1 nM–100 µM of acetylcholine (ACh) to initiate relaxation. B: maximal tension in response to 1 µM PE perfusion in aortic rings treated with vehicle or TMAO. C: percentage of relaxation after perfusion with 10 nM-100 µM ACh in aortic rings treated with vehicle, TMAO, or N^G-nitro-l-arginine methyl ester (l-NAME; 100 µM). *P < 0.05, statistical difference from l-NAME (P < 0.05). TMAO, 300 µM.