4/14/2021 0 Comments Download Pca Col 4.5
NOTE: Usually, it is necessary to add a significant amount of KOH to reach a pH of 7.1. Place the volumetric balloon in a box filled with ice to cool down the solution to 15 C.This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.
These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca 2 )-containing solutions to determine, amongst others: maximum force development, myofilament Ca 2 -sensitivity (pCa 50 ), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. ![]() Multicellular cardiac muscles strips include a heterogeneous population of cells, including contractile cardiomyocytes with an unknown pattern of orientation and force generation, electrical activity and stressstrain distributions as well as a surrounding connective tissue matrix 3, 4. A preparation without collagen and containing a single cardiomyocyte would allow measurement of sarcomere length and cross-bridge contractile properties in a very precise and controlled manner 5, 6. Therefore, over the last four decades, several methodologies were developed allowing investigating the mechanical, contractile, and relaxation properties of a single cardiomyocyte 6, 7. The contractile function of these cells is strongly dependent on sarcomere length and cross-bridge cycling kinetics 3. Thus, it is desirable to investigate muscle function directly in single isolated cardiac cells, considering that it allows assessing sarcomere length and performance as well as cross-bridge function and contractile properties. However, isolating and attaching functional cardiomyocytes with a reasonable optical sarcomere resolution while recording force measurement at the N level is still challenging and evolving 3, 6. Other challenges are the logistics that need to be installed to isolate cardiomyocytes from freshly collected biopsies. The unpredictability of human biopsies collection, for instance, may jeopardize the feasibility of the experiments. Indeed a progressive refinement of methodologies to assess cardiac function in vitro on a smaller level of complexity allows proper integration of the results to the whole body and translate them to the clinical scenario 7. Altogether, using samples stored at -80 C to extract cardiomyocytes may be an appealing alternative. The result of this homogenization is a suspension of skinned bundled and isolated cells with varying degrees of sarcolemmal damage, wherein the myoplasm is exposed to the bathing medium and all the cellular components are washed out. Structures such as the myofibrils that are further away from the sarcolemma are preserved. Thus, sarcomere shortening and functional properties associated with the myofibrillar apparatus are kept intact and can be recorded 8, 9. A permeabilized, or skinned, cardiomyocyte is placed in an experimental chamber containing a relaxing solution (Ca 2 2 concentrations, the determination of actin-myosin cross-bridge kinetics and the measurement of the passive tension of the mounted cardiomyocytes at pre-defined sarcomere lengths ( Figure 1 ). The competent local authorities approved this experimental protocol (018833). Dissolve the reagent above in 500 mL and adjust the pH to 7.0 with KOH. Adjust the final volume to 1000 mL. ![]() Put the volumetric balloon in a box with ice to cool down the solution to 15 C. Agitate this solution continuously with a magnetic stirrer until the moment of mixing it with the relaxing solution. NOTE: Usually, it is necessary to add a significant amount of KOH to reach a pH of 7.1. Place the volumetric balloon in a box filled with ice to cool down the solution to 15 C.
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