Impaired cellular contractile function in thiamine‐deficient rat cardiomyocytes

Background Thiamine (vitamin B1) is an essential cofactor of important metabolic enzymes. 1,2 Studies have demonstrated that thiamine deficiency (TD) leads to changes in cardiac morphological, electrophysiological and mechanical properties, as well as in cardiac metabolism. 3 - 9 Clinical studies have indicated that the use of loop diuretics can induce TD and eventually cause heart dysfunction. The most important reported clinical manifestation of TD involves high-output heart failure. In humans, TD damages the cardiovascular system causing heart failure. 10-12 The altered excitation-contraction coupling that characterizes cardiac failure occurs due to changes in Ca 2+ dynamics, action potential duration, global Ca 2+ transient, and contraction and relaxation rates. 13 Oliveira et al. 8 found that thiamine deprivation decreased the global Ca 2+ release from the sarcoplasmic reticulum (SR) due to the lower SR Ca 2+ load without changes in Ca 2+ current density. Aim According to data in the literature, TD may elicit deregulation of SR Ca 2+ handling in the heart, with implications on cellular cardiac contractility. However, it is unknown whether TD would cause contractile dysfunction at the cellular level. In this study, we sought to determine if TD affects cellular contractility. Methods Male Wistar rats (200-250 g) were randomly allocated to receive a diet containing thiamine (control, n = 6) or a TD diet (TD, n = 6) for 35 days as described previously by our group. 8,9 All experimental procedures were conducted in accordance with international guidelines (Declaration of Helsinki). Cell isolation Ventricular cardiomyocytes from age-matched control and TD rats were enzymatically isolated as described previously. 8,9 Briefly, the heart was mounted on a custom-designed Langendorff system, perfused for ∼3-5 min with calcium-free solution following perfusion with 1 mg/mL collagenase type II (Worthington, USA). After 10-15 min, single cells were isolated by mechanical dispersion and stored in Dulbecco's modified Eagle's medium. The average cell yield under these conditions is 60-70%. Measurement of cellular contractility Cellular contractility was measured as described previously. 14 Briefly, isolated cells were placed in an experimental chamber with a glass cover-slip base mounted on the stage of an inverted microscope. Cells were perfused with Tyrodes solution containing 1.8 mM CaCl 2 and field stimulated at frequencies of 1 and 3 Hz (20 V, 5 ms duration square pulses). Cells were imaged using a NTSC camera in a partial scanning mode. Cell shortening in response to electrical stimulation was measured with a video-edge detection system at 240 Hz frame rate (lonoptix). All parameters were evaluated using customized software developed in MatLab ® platform. 14 Experiments were performed at room temperature (∼25°C). Only calcium-tolerant, quiescent, rod-shaped myocytes showing clear cross striations were studied. Statistical analysis All results are expressed as mean ± standard error of the mean. For statistical analysis, we used Student's t-test and ANOVA for repeated measurements with Bonferroni post hoc testing. Values of P < 0.05 were considered significant. Results After 35 days of thiamine deprivation, cellular ATP availability decreases, leading to impairment of heart contraction as we reported in a previous study. 8 Thus, we hypothesized that a reduction in circulating thiamine levels would affect cardiomyocyte.