Substrate Stiffness Affects the Functional Maturation of Neonatal Rat Ventricular Myocytes
Jeffrey G. Jacot 1*, Andrew D. McCulloch 2 and Jeffrey H. Omens 3
1 University of California, San Diego
2 Univer. of California, San Diego
3 Univer. of California, San Diego
* To whom correspondence should be addressed. E-mail: jjacot{at}ucsd.edu.
Submitted on October 31, 2007
Revised on January 14, 2008
Accepted on 12 May 2008
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Abstract |
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Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. In order to investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes (NRVMs) for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10 kPa substrates developed aligned sarcomeres, while cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10 kPa, than on stiffer or softer substrates. Cardiomyocytes on 10 kPa gels also had the largest calcium transients, sarcoplasmic calcium stores and SERCA2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocytes maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase (ROCK) and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances NRVM maturation. Deviations from ideal stiffness result in lower expression of SERCA, less stored calcium, smaller calcium transients and lower force. On very stiff substrates, this adaptation appears to involve ROCK.
Key Words:
Calcium, Elastic Modulus, Polyacrylamide, ROCK, RhoA Kinase, Traction Force
