Taurine Renders the Cell Resistant to Ischemia Induced Injury in Culture Neonatal Rat Cardiomyocytes

Summary

The objective of this study was to determine the effects of taurine, a beta-amino acid, on mammalian heart cells, specifically neonatal rat cardiomyocytes. Taurine is found in high concentrations in the heart and is responsible for several important functions. For example, the article states that taurine is associated with osmoregulation, ion transport, protein phosphorylation, cell signaling, and energy metabolism.

When cardiomyocytes are subjected to ischemic conditions, the toxic levels in the cell rise and the taurine levels in the cell drastically drop. The authors believe that this reduction of taurine concentration can result in a net decrease in osmolarity, a disruption in protein phosphorylation, and a greater risk of calcium overload. Due to the importance of taurine in a cardiomyocyte’s viability and functional capacity, this study wished to examine the cellular response of cardiomyocytes within an ischemic environment and the effects of exogenously introduced taurine in the system.

For this study, the authors had chosen neonatal rat cardiomyocytes. This cell choice allowed researchers to control the concentration of taurine within the cell. Immature animals do not biosynthesize enough taurine and must depend on an external source. Due to this, the taurine measured would predominantly be the taurine added. These cells were then placed in ischemic-conditioned flasks filled with varying levels of taurine. Ischemia was replicated through a combination of media, phosphate-buffered saline, and bubbled with a CO₂-N₂ mixture within a sealed flask. The environment inside the flask had an initial pH of 7.4 and was depleted of oxygen. These conditions ensured that the cells were experiencing hypoxia, acidosis, and stagnant medium. A control was made to run alongside these samples and the media was frequently changed within the flask.

The intracellular taurine content was measured using high-performance liquid chromatography. The researchers had found that by subjecting cardiomyocytes to a 20 mM concentration of taurine within ischemic conditions, taurine concentrations became elevated, surpassing the initial amount of taurine added.

Also, the cellular morphology and beating status of the cardiomyocytes were evaluated with an inverted phase contrast microscope and videomonitor. Morphological degeneration of the cell was seen by the authors to become lessened in the flask with taurine, as seen in Figure 1. After 72 hours of simulated ischemia, 87% of the untreated cells were degenerated and only 61% of the taurine-treated cells were degenerated. Cell apoptosis were also reduced with the introduction of exogenous taurine. In the same time span the addition of taurine nearly halved the percentage of apoptotic cells found in an ischemic environment.

In addition, the creatine phosphokinase content was determined using a CPL-test Wako kit and bovine serum albumin standards. Taurine was seen to reduce the amount of loss creatine phosphokinase due to ischemia as well as help in the retention of a cell’s beating function. There was nearly three times as much CPK activity in taurine-treated cells than untreated cells after 72 hours and there was a quadrupled in percentage of beating cells. Taurine did not help in intracellular ATP reduction.

They used genomic DNA electrophoresis to determine if the cardiomyocytes were in the process of apoptosis. Hypoxia was explained to induce DNA fragmentation in the cell, as seen in Figure 2. Taurine seemed to reduce the amount of fragmented DNA in the cardiomyocytes.

Finally, a western blot was used to measure the amounts of Bcl-2, p53, and Bax within the cell lysates. These three molecules are apoptotic factors that are indicators of apoptosis. Figure 4 shows that Bel-2, an anti-apoptotic factor, increased by 125% while Figure 5 shows that p53, an apoptotic promoter, decreased by 16%. Bax, a proapoptotic factor, was not affected by this taurine addition.

In this study, the authors were able to form conclusions on the effects of taurine to cardiomyocytes. They were able to deduce that taurine does not affect the time scale of cell death, but alternatively, it inhibits apoptosis, which occurs at a later stage of cell death later, more than necrosis, which occurs earlier. Furthermore, the researchers formulated three theories on why taurine has such an effect on cells experiencing ischemic conditions. The first is that taurine might be downregulating p53. Second, taurine might be interfering with caspases activation. The last theory is that taurine might be promoting Bcl-2 expression.

Significance

Taurine shows promise as a significant amino acid in therapeutic use. Taurine’s ability to block apoptosis has great potential in restoring hearts that are failing. Taurine has already been incorporated in the medical field to treat patients with heart failure. It is also believed to partially prevent damage to the heart caused by fatal conditions such as doxorubicin toxicity, aortic regurgitation, and calcium overload. In time, a definitive pathway may emerge for taurine and through this information, better control of taurine administration could be achieved. Also, it may also be useful in other tissues around the body that are experiencing ischemia or other forms of apoptotic conditions.

Critique

This journal article was thoroughly written and contained a methodical procedure and analysis section. My main concern for this study though is their investigation of apoptosis through the examination of fragmented genomic DNA. I feel that this section was not explained enough. They did not mention the mechanism of how the DNA is fragmented. Furthermore, the test seems to be a bit unnecessary. They were able to quantify apoptosis through other methods and this qualitative test (DNA electrophoresis) does not give any further significant information on the ischemic/taurine effects on the cell line.