A Novel Method of Isolating Myofibrils From Primary Cardiomyocyte Culture Suitable for Myofibril Mechanical Study

Figures

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  Figure 1. (A) Experimental scheme: hearts from Sprague Dawley rats are retrograde perfused. Before enzymatic digestion, a small section of the apex is cut to obtain myofibrils by skinning in Triton-X100. Remaining heart was digested in Liberase DH to obtained primary ARVM culture. Myofibrils from ARVM culture were obtained by sucrose based osmotic shock method. (B) Representative images of 10x magnification demonstrating the different quantity and quality of myofibrils isolated using the different techniques. Black arrows indicate useable myofibrils in each field. The myocyte fragments shown in the panel of ARVMs harvested using Triton-x show a morphology distinct from the fragments of the other harvest techniques. (C) Representative image of a myofibril isolated from ARVMs using the sucrose-based method. (D) Representative trace from an ARMV-derived myofibril activated and relaxed by fast solution switching. ARVM, adult rat ventricular myocyte.

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  Figure 2. (A) Force-pCa relationship of myofibrils obtained from cardiomyocyte culture day 1 (blue), culture day 3 (red) vs. myofibrils isolated from cardiac tissue (black). At each pCa, maximal tension was normalized to tension generated at pCa 4.5 (P0). Data points are mean ± SEM. Lines are drawn according to the parameters estimated by fitting to the Hill equation (P/ P0 = 1/)(1 +10(−nh(pCa50−pCa))). N = 4 rats, 16–20 myofibrils. (B) Force-pCa relationship of myofibrils obtained from ARVMs that were PE-treated (black), myofibrils treated with PE and the β-blocker propranolol (BB) (red) vs. vehicle treated control (blue). N = 6 rats, 16–22 myofibrils. ARVM, adult rat ventricular myocyte; PE, phenylephrine; BB, β-blocker propranolol.

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  Figure 3. (A) Myofilament protein phosphorylation from ARVMs treated with PE for 2 and 48 h. ProQ Diamond® stain (PQD) was used to determine the global phosphorylation status of myofilament proteins. The same gel was stained with commassie brilliant blue (CBB) to determine relative abundance of proteins. (B) Quantification of PQD over CBB densitometries. Only those sarcomeric proteins with significant change in global phosphorylation are shown. Phosphorylation of TnI is significantly increased in ARVMs after 2 h (p < 0.0001) and 48 h (p = 0.003) of PE treatment. Myosin binding protein C (MyBP-C) trends to increased phosphorylation at 48 h of treatment (p = 0.056) but not at 2 h. N = 5 rats. ARVM, adult rat ventricular myocyte; PE, phenylephrine; CBB, commassie brilliant blue; TnI, troponin I; MyBP-C, myosin binding protein C.

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  Figure 4. (A) Representative Western blot demonstrating serine 23/24 is preferentially phosphorylated with PE treatment. Each lane is protein isolated from different experiments. These conditions were repeated in ARVMs cultured from 5 different rat hearts. (B) Representative image of a Western blot showing pre-treatment of ARVMs with the β-blocker propranolol (BB) leadsto inhibition of TnI phosphorylation at serine 23/24. Experiment was repeated in ARVMs cultured from 3 different rat hearts. (C) Quantification of TnI serine 23/24 phosphorylation normalized to total TnI. PE significantly increases phosphorylation of TnI at Ser 23/24 (p < 0.001). Treatment with PE and a β-blocker (BB) propranolol, normalizes TnI phosphorylation (PE tx v PE+BB p < 0.001). Each lane is protein isolated from different experiments. These conditions were repeated in ARVMs cultured from 3 different rat hearts. TnI, troponin I; PE, phenylephrine; ARVM, adult rat ventricular myocyte; BB, β-blocker propranolol.