Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes

Understanding intracellular calcium (Ca) regulation is essential for modeling cardiac excitation-contraction coupling. Prior research has shown that calcium buffering involves multiple intracellular compartments, including the sarcoplasmic reticulum (SR) and mitochondria. However, the specific contribution of passive calcium binding and its impact on SR uptake remains unclear. This gap motivated researchers to investigate the passive buffering capacity and SR uptake in permeabilized cardiac myocytes. No prior work had resolved the exact parameters of passive Ca binding in these cells. Existing models rely on data from earlier studies, which may not fully capture the complexity of calcium dynamics in cardiac cells. The discrepancy between theoretical expectations and observed values suggests a need for updated measurements. Researchers have proposed that passive Ca binding may play a larger role than previously assumed. This uncertainty drives the need for direct experimental quantification of buffering parameters in permeabilized myocytes.

The aim of this study was to measure passive calcium binding and SR uptake in permeabilized rabbit ventricular myocytes. The specific problem addressed is the lack of precise data on how much calcium is passively bound in these cells and how this affects SR uptake. The motivation stems from the need to refine models of calcium regulation in cardiac cells. The researchers sought to determine the buffering capacity of myocytes under conditions that prevent SR or mitochondrial uptake. By measuring calcium binding directly, they aimed to clarify the role of passive buffering in cardiac calcium homeostasis. The study also aimed to compare calcium measurements obtained using different methods. The findings could help improve the accuracy of computational models of calcium signaling. This work contributes to a better understanding of how calcium is regulated in cardiac muscle.

The researchers used permeabilized rabbit ventricular myocytes to study calcium binding and uptake. They measured passive calcium binding using a calcium-selective minielectrode in suspensions equilibrated with thapsigargin and ruthenium red. These agents were used to prevent SR and mitochondrial calcium uptake. Calcium binding was determined by titrating the myocytes with calcium and subtracting the values from a blank titration. The data were fitted to a Michaelis binding curve with two binding sites. The researchers also measured SR calcium uptake in the presence of ruthenium red. They compared calcium concentration changes using a calcium electrode and indo-1 fluorescence. Oxalate was used to assess its effect on calcium uptake and buffering. The experimental setup allowed for precise quantification of passive calcium binding and SR uptake.

Passive calcium binding was described by a Michaelis binding curve with two sites: K1 = 0.42 microM and n1 = 1.27 nmol/mg cell protein, and K2 = 79 microM and n2 = 4.13 nmol/mg cell protein. The passive calcium buffering was approximately twice the value expected from prior studies. The maximal SR calcium uptake in the presence of ruthenium red was 5.16 nmol/mg cell protein with a K 1/2 of 1.0 microM. In the absence of ruthenium red, a significant calcium uptake attributed to mitochondria was observed between 10 and 100 microM free calcium. Rapid changes in free calcium concentration measured with a calcium electrode were slower than those measured with indo-1. Oxalate increased the uptake rate and eliminated the difference in calcium measurements. This suggests the presence of spatial calcium gradients in permeabilized myocytes. The new estimates of calcium buffering should improve models of cardiac calcium regulation.

The study provides new estimates of passive calcium buffering and SR uptake in permeabilized cardiac myocytes. The authors suggest that passive calcium binding may be more significant than previously assumed. The findings indicate that calcium buffering in cardiac cells is higher than expected from earlier studies. The researchers propose that spatial gradients of free calcium exist in permeabilized myocytes without calcium buffering. Oxalate was shown to increase uptake rates and eliminate differences in calcium measurements. The results highlight the importance of considering passive buffering in models of calcium regulation. The authors suggest that these findings could improve the accuracy of computational models of cardiac function. The study contributes to a better understanding of calcium dynamics in cardiac muscle.