A nanophysiometer to study force – excitation coupling in single cardiac myocytes

The dissertation describes the design and application of the Nanophysiometer, a microfluidic network that is combined with a thin film microelectrode array to study force – excitation coupling in single cardiac myocytes. The microfluidic device was fabricated in a silicone elastomer and imaged using an inverted microscope, a high-speed CCD camera and an optical fiber array coupled to photomultipliers for high-bandwidth fluorescence recordings. The Nanophysiometer automatically aligned and stabilized single cardiac myocytes on the microscope during long-term sarcomere contraction measurements, thereby reducing motion artifacts. Measurements of intracellular calcium concentration and sarcomere length were combined to test the hypothesis that phospholamban (PLN) ablation in mice increases the calcium sensitivity of the cardiac myofilaments. It was demonstrated that sarcomere acceleration may be used as an index of contractility when the length-dependent passive resistance and the velocity-dependent viscous damping force are considered. A previously published proteomic analysis of PLN deficient mouse hearts and our results indicate that significant changes in myofilament protein expression and phosphorylation must have contributed to the increased force development in hearts lacking phospholamban. Further more, genetic phospholamban deletion significantly reduced the dependence of the sarcoplasmic reticulum (SR) Ca++ uptake on the stimulation frequency, but did not abolish it. Even the combination of genetic deletion of PLN and chronic inhibition of the Ca++/Calmodulin dependent protein kinase II (CaMKII) did not prevent the frequency dependence of the SR Ca++ uptake. Our results suggest that in the mammalian ventricle, a mechanism of frequency adaptation must exist that does not require PLN or CaMKII. However, chronic CaMKII inhibition significantly slowed Ca++ release at physiological frequencies in the absence of PLN indicating a role of CaMKII in the regulation of SR Ca++ release at high SR Ca++ load. When the techniques described here are combined, the Nanophysiometer will be capable of simultaneous measurements of extracellular potentials, intracellular and extracellular ion and metabolite concentrations (e.g. Ca++, pH, O2) and sarcomere length in a chemically controlled microfluidic environment.