Modulation of Cardiac Fibroblast to Myofibroblast Transition by Rho-Associated Kinases ROCK1 and ROCK2

by Svenja Hartmann
Doctoral thesis
Date of Examination:2016-10-18
Date of issue:2016-10-25
Advisor:Prof. Dr. Susanne Lutz
Referee:Prof. Dr. Susanne Lutz
Referee:Prof. Dr. Thomas Meyer
Persistent Address: http://dx.doi.org/10.53846/goediss-5931

 

 

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Abstract

English

Rho-associated kinases ROCK1 and ROCK2 are serine/threonine kinases that are downstream targets of the small GTPases RhoA, RhoB, and RhoC. They play a critical role in the pathogenesis of myocardial fibrosis; however, their specific function in cardiac fibroblasts (cFB) remains unclear. Remodelling processes in the diseased heart propels the transition of cFBs to a myofibroblast phenotype exemplified by increased proliferation, migration and synthesis of extracellular matrix (ECM) proteins. Therefore, we sought to investigate whether ROCK1 and ROCK2 protein signalling intermediates have an impact on cFB characteristics in isolated cells and engineered cardiac tissue. Knockdown of ROCK1 and ROCK2 and inhibition of ROCK1/2 activity altered cFB morphology, disrupted cytoskeletal structures, and increased adhesion velocity. In line, absolute number and area of focal adhesions was increased in ROCK1 and ROCK2 knockdown cells. Moreover, the double knockdown and inhibition of ROCK1/2 attenuated proliferation of cFBs. Interestingly, downregulation of either ROCK1 or ROCK2 decreased migration velocity and absolute distance, whereas the double knockdown and inhibition of ROCK activity increased both. Destructive tensile strength measurement of a three-dimensional engineered tissue model composed of cFBs (engineered connective tissue, ECT) treated with ROCK inhibitors showed that rigidity was significantly reduced compared to control tissues. This suggests that ROCK1 and ROCK2 influence the regulation and turnover of the ECM and thus viscoelastic properties of engineered tissues. Indeed, qPCR analysis of ROCK inhibitor-treated ECT showed that both ROCKs are involved in the regulation of several ECM proteins, including elastin and its degrading enzyme MMP12. In contraction measurements, human engineered heart muscle (hEHM) treated with ROCK inhibitors developed a significantly lower force of contraction per cross sectional area than control hEHM, whereas in the rat model (rEHM) no change in force of contraction could be detected. This demonstrates that ROCKs influence contractile parameters of engineered tissues.
Keywords: Cardiac Fibroblast; Myofibroblast; Rho-associated kinase; ROCK1 and ROCK2; Cardiac Fibrosis; Engineered tissue
 
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