Figure S4.
Validating GFP-NaV1.5 and EB1-mRFP as surrogates for native NaV1.5 and EB1 in ventricular myocytes. Validating GFP-NaV1.5 and EB1-mRFP as surrogates for native NaV1.5 and EB1 in ventricular myocytes. (A) Myocytes were incubated without (no Adv) or with Adv-GFP-NaV1.5 for 12 h. The medium was exchanged for virus-free fresh medium and culture continued for 0, 6, 20, 36, or 48 h (total culture times: 12, 18, 32, 48, and 60 h). At specified times, myocytes were fixed for experiments. Top: Airyscan images of GFP-NaV1.5 immunofluorescence detected with GFP goat Ab (culture time: 18–48 h), and GFP fluorescence (FP) of a myocyte cultured for 60 h. Right: Immunofluorescence of native NaV1.5 in a freshly isolated myocyte. At 18 h, GFP-NaV1.5 clustered around nuclear envelope representing protein translation in the NE/rER. At 24 h, GFP-NaV1.5 spread out in striations along z-lines, similar to the nuclear envelope to SR along t-tubules or NEST pathway (He et al., 2020). By 32 h, GFP-NaV1.5 reached the lateral surface and ICD. The distribution pattern was stable at 48 and 60 h culture times. These data showed that the steady-state distribution pattern of GFP-NaV1.5 was similar to that of native NaV1.5, except active GFP-NaV1.5 translation at NE/rER. Bottom: Degree of GFP-NaV1.5 expression versus native NaV1.5 based on pixel contents. Myocytes without or with Adv-GFP-NaV1.5 for the total culture times shown along the abscissa were immunostained with Alomone asc005 rabbit Ab, which detected both GFP-NaV1.5 and native NaV1.5. The total pixel contents were quantified and normalized to the mean pixel contents of “No Adv” myocytes of the same culture time. Shown is a bar graph of mean + SE superimposed with individual data points. “Adv-GFP-NaV1.5” myocytes did not have more immunofluorescence than “no Adv” myocytes until the 32 h time point. By the 48 h time point, “Adv-GFP-NaV1.5” myocytes had 30% more immunofluorescence than “no Adv” myocytes. These data showed a modest expression of GFP-NaV1.5 over native NaV1.5. The native NaV1.5 immunofluorescence image is a duplicate from Fig. 3 A, NaV1.5 immunofluorescence image from a control myocyte after culture for 1 h. (B) (a) Confirming immunoblot banding pattern of EB1-mRFP and native EB1 in HEK293 cells. EB1 rat Ab detected only the native EB1 band (30 kD, blue dot) in untransfected cells, while it detected both native EB1 and EB1-mRFP (expected 55.7 kD, two bands at and above 50 kD, red star) in transfected cells. The identity of the two EB1-mRFP bands was confirmed by reprobing the membrane with mCherry rabbit Ab. (b) EB1-mRFP expressed in ventricular myocytes. Left: IP lane shows EB1-mRFP coimmunoprecipitation with native EB1 (red star and blue circle), indicating dimerization between the two to form active EB1 (Chen et al., 2014). The WCL lane shows that Adv-mediated EB1-mRFP expression was ∼150% over native EB1 (band intensity ratio 2.5:1). This immunoblot image is modified from the same experiment shown in Fig. 9 B, bottom right. Right: In live myocytes (images obtained during FRAP experiments, Fig. 9 E), EB1-mRFP had a wavy strand morphology along the long axis of CON myocyte, suggesting binding along the microtubule lattice. In PKA myocyte, EB1-mRFP clustered to ICD and manifested striations at the z-plane adjacent to myocyte surface. The difference in EB1-mRFP patterns between CON and PKA myocytes is similar that of native EB1 between CON and PKA myocytes (Figs. 4 and 8). Scale bars are 10 mm in A and 5 mm in B. Source data are available for this figure: SourceData FS4.

Validating GFP-Na V 1.5 and EB1-mRFP as surrogates for native Na V 1.5 and EB1 in ventricular myocytes. Validating GFP-NaV1.5 and EB1-mRFP as surrogates for native NaV1.5 and EB1 in ventricular myocytes. (A) Myocytes were incubated without (no Adv) or with Adv-GFP-NaV1.5 for 12 h. The medium was exchanged for virus-free fresh medium and culture continued for 0, 6, 20, 36, or 48 h (total culture times: 12, 18, 32, 48, and 60 h). At specified times, myocytes were fixed for experiments. Top: Airyscan images of GFP-NaV1.5 immunofluorescence detected with GFP goat Ab (culture time: 18–48 h), and GFP fluorescence (FP) of a myocyte cultured for 60 h. Right: Immunofluorescence of native NaV1.5 in a freshly isolated myocyte. At 18 h, GFP-NaV1.5 clustered around nuclear envelope representing protein translation in the NE/rER. At 24 h, GFP-NaV1.5 spread out in striations along z-lines, similar to the nuclear envelope to SR along t-tubules or NEST pathway (He et al., 2020). By 32 h, GFP-NaV1.5 reached the lateral surface and ICD. The distribution pattern was stable at 48 and 60 h culture times. These data showed that the steady-state distribution pattern of GFP-NaV1.5 was similar to that of native NaV1.5, except active GFP-NaV1.5 translation at NE/rER. Bottom: Degree of GFP-NaV1.5 expression versus native NaV1.5 based on pixel contents. Myocytes without or with Adv-GFP-NaV1.5 for the total culture times shown along the abscissa were immunostained with Alomone asc005 rabbit Ab, which detected both GFP-NaV1.5 and native NaV1.5. The total pixel contents were quantified and normalized to the mean pixel contents of “No Adv” myocytes of the same culture time. Shown is a bar graph of mean + SE superimposed with individual data points. “Adv-GFP-NaV1.5” myocytes did not have more immunofluorescence than “no Adv” myocytes until the 32 h time point. By the 48 h time point, “Adv-GFP-NaV1.5” myocytes had 30% more immunofluorescence than “no Adv” myocytes. These data showed a modest expression of GFP-NaV1.5 over native NaV1.5. The native NaV1.5 immunofluorescence image is a duplicate from Fig. 3 A, NaV1.5 immunofluorescence image from a control myocyte after culture for 1 h. (B) (a) Confirming immunoblot banding pattern of EB1-mRFP and native EB1 in HEK293 cells. EB1 rat Ab detected only the native EB1 band (30 kD, blue dot) in untransfected cells, while it detected both native EB1 and EB1-mRFP (expected 55.7 kD, two bands at and above 50 kD, red star) in transfected cells. The identity of the two EB1-mRFP bands was confirmed by reprobing the membrane with mCherry rabbit Ab. (b) EB1-mRFP expressed in ventricular myocytes. Left: IP lane shows EB1-mRFP coimmunoprecipitation with native EB1 (red star and blue circle), indicating dimerization between the two to form active EB1 (Chen et al., 2014). The WCL lane shows that Adv-mediated EB1-mRFP expression was ∼150% over native EB1 (band intensity ratio 2.5:1). This immunoblot image is modified from the same experiment shown in Fig. 9 B, bottom right. Right: In live myocytes (images obtained during FRAP experiments, Fig. 9 E), EB1-mRFP had a wavy strand morphology along the long axis of CON myocyte, suggesting binding along the microtubule lattice. In PKA myocyte, EB1-mRFP clustered to ICD and manifested striations at the z-plane adjacent to myocyte surface. The difference in EB1-mRFP patterns between CON and PKA myocytes is similar that of native EB1 between CON and PKA myocytes (Figs. 4 and 8). Scale bars are 10 mm in A and 5 mm in B. Source data are available for this figure: SourceData FS4.

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