T‐tubule recovery after detubulation in isolated mouse cardiomyocytes

Figures

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  FIGURE 1. Dynamics of trapped dextran fluorescence after hyposmotic detubulation. (a) Protocol for characterizing the time and temperature dependences of trapped dextran fluorescence after detubulation. Cardiomyocytes were detubulated at room temperature in the presence of extracellular 3 kDa dextran by transient application of hyposmotic 0.6 Na Tyrode. Afterwards, cardiomyocytes were incubated at the indicated temperatures for 0–120 min before imaging. (b) Representative images of cardiomyocytes with trapped dextran following incubation at 32°C for 0, 30, or 60 min after detubulation with hyposmotic Tyrode. Scale bar: 20 μm. (c) Quantification of dextran fluorescence after detubulation. There were no significant changes in dextran fluorescence when cardiomyocytes were incubated on ice (linear fit; gray line). Time‐dependent decline in dextran fluorescence was characterized by single exponential approximation (gray lines): F = A*exp(−t/τ) + C. At 25°C and 32°C the time course of dextran fluorescence was characterized by time constants τ h 25 = 42.7 ± 5.9 min and τ h 32 = 19.8 ± 3.8 min, respectively. Each point represents the average fluorescence from n = 20–30 cells from 2 to 3 separate…

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  FIGURE 2. Recovery of diffusional properties of t‐tubules after hyposmotic detubulation. (a) Representative time courses of diffusion of t‐tubular dextran recorded in normal cells (Control), immediately after detubulation (0 min), or after detubulation followed by 1 h (60 min) incubation at 32°C. ▼‐ indicates the amplitude of t‐tubular dextran fluorescence. (b) Quantification of changes in fluorescence amplitude (reflects the volume of the t‐tubule network that is accessible to extracellular solution). Relative amplitude was not significantly different for control and 60 min conditions. (c) Quantification of changes in diffusion time constant of 3 kDa dextran (reflects diffusional accessibility of the t‐tubule network, in particular the magnitude of t‐tubular dilations and constrictions). n = 12, 15, and 17 cells from three separate preparations for control, 0 min, and 60 min, respectively.

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  FIGURE 3. Electrophysiological characterization of diffusional properties of t‐tubules after hyposmotic detubulation. (a) Representative traces of I K1,tail currents normalized to I K,end (open circle) recorded from normal cells (Control), immediately after detubulation (0 min), or after detubulation followed by 1 h incubation at 32°C (60 min). Inset: the origin of I K1,tail current. (b) Quantification of I N K1,tail current amplitudes. (c) Quantification of I K1,tail time constant. (d) Quantification of membrane capacitance, Cm. n = 10–12 cells, for each category.

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  FIGURE 4. Effects of microtubule‐modifying agents. (a) Protocol for the application of pharmaceutical agents (X) using hyposmotically‐induced detubulation approach. (b) Effects of various microtubule‐modifying agents on the amount of trapped dextran immediately after detubulation (0 min) or 15 min after detubulation. Microtubule modifiers: disruptors colchicine (Col) and nocodazole (Noc), and stabilizer taxol (Tax). None of the agents had a significant effect on the initial amount of trapped dextran (0 min). However, both disruption and stabilization of microtubules led to an increased amount of trapped dextran after 15 min post‐detubulation. n = 200 (Control), 37–90 (10 μM Col), 20 (1 μM Tax), 34–35 (10 μM Tax), and 30 (10 μM Noc).

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  FIGURE 5. Effects of metabolic inhibition and Ca2+ manipulation. (a) Protocol for hyposmotic detubulation. (b) Quantification of the effects of FCCP (uncoupler of mitochondrial oxidative phosphorylation; 25 μM) on the post‐detubulation dextran fluorescence. FCCP was added immediately after detubulation. n = 25–30 cells. (c) Quantification of the effect of EGTA (1 mM) and nicardipine (10 μM) on the fluorescence of trapped dextran. EGTA and nicardipine were added during hyposmotic stress or after detubulation. Nicardipine was removed after incubation, but cells exposed to EGTA were maintained in EGTA containing solution to avoid unwanted effects due to calcium reintroduction. n = 20–30 cells.