Novel method of obtaining myofibrils from cardiomyocyte cultures

Myofibril based mechanical studies allow targeted evaluation of sarcomeric protein function. Here we describe a novel method of obtaining myofibrils from primary cardiomyocyte culture. Adult rat ventricular myocytes (ARVMs) were obtained by enzymatic digestion and maintained in serum free environment for 72 h. ARVMs were homogenized in relaxing solution (pCa 9.0) with 20 % sucrose and myofibril suspension was made. Myofibrils, mounted in a force recording apparatus, were Ca2+-activated and fully relaxed by fast solution switching at 15 C and 2.1 lm initial sarcomere length. Results from ARVMs myofibrils were compared to those of myofibrils obtained from skinned ventricular tissue (0.05 % Triton-X100). At maximal Ca2+-activation (pCa 4.5) myofibril mechanical parameters from ARVMs were 8 mN/mm2 (resting tension), 156.4 mN/ mm2 (maximal active tension, Po), 5.4 s-1 (rate of force activation, kACT), 5.2 s-1 (rate of force redevelopment following releaserestretch, kTR), 41.6 ms (linear relaxation duration), and 13.6 s-1 (exponential relaxation rate). The parameters of the myofibrils isolated from the ventricular tissue were not significantly different. Degree of run-down (decrease in Po and kACT between subsequent activation-relaxation cycles) was evaluated. Between first and third activations, we noted the following % changes in myofibrils from ARVM cultures: -23.6 % (Po), -29.6 % (kACT). The magnitude of run-down was not statistically different between myofibrils from ARVMs versus the traditional Triton skinned samples. Finally, pCa50 from Triton skinned, ARVM culture day 0, and ARVM culture day 3 myofibrils were 5.76, 5.74, and 5.71, respectively. Myofibrils obtained from ARVMs is a viable method to study myofibril mechanics. This culture based approach to obtaining myofibrils will allow pharmacological and genetic manipulation of the cardiomyocytes to correlate biochemical and biophysical properties.