Quantitative tests reveal that microtubules tune the healthy heart but underlie arrhythmias in pathology

Figures

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  Figure 1.. Simplified diagram of stretch-dependent Ca2+ signalling in heart [Ca2+]i, cytosolic [Ca2+]; CaMKII, Ca2+/calmodulin-dependent protein kinase II; MT, microtubule; nNOS, neuronal nitric oxide synthase; NOX2, NADPH oxidase 2; RNS, reactive nitrogen species; ROS, reactive oxygen species; RyR2, ryanodine receptor, type 2 SAC, stretch-activated channel.

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  Figure 2.. Microtubules in heart A and B, VCMs from WT (A) and MDX (B) hearts immunolabelled for α-tubulin. C, quantification of MT density was obtained by normalizing the skeletonized binary area of α-tubulin (A and B) over the binary area of actin (not shown). D, western blots for free and polymerized fraction of a-tubulin and glu-tubulin, respectively. E, quantification of western blot in MDX hearts showing the overexpression of tubulin proteins over WT hearts. F, western blots and quantification (fold change) for total gp91 expression in WT and MDX. ****P < 0.001, *P < 0.05 over WT group.

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  Figure 3.. Physiological stretch alters Ca2+ entry in heart VCMs are first attached to glass cardiac fingers (see Methods). VCMs are loaded with Fura-2 AM (5 μM, 15 min). A, extracellular Ca2+ (1.8 mM) is replaced with equimolar Mn2+ which enters the cell via sarcolemmal Ca2+ channels and quenches Fura-2 florescence (see Slope 1). During a 2 Hz cyclic stretch, elevated Ca2+ influx increases the quench rate (see Slope 2). B, analysis of Ca2+ influx rates. Statistics: MDX compared to WT (*P < 0.05), Stretch compared to Rest (#P < 0.05), and NAC compared to vehicle (§P < 0.05).

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  Figure 4.. Stretch-dependent changes in Ca2+ signalling in heart A and B, representative, longitudinal confocal linescans for WT (A) and MDX (B) VCMs loaded with Fluo-4 AM. C, Ca2+ spark frequency, amplitude, FDHM and FWHM before, during and after a static, 8% stretch. Statistics: Stretch compared to Before (*P < 0.05).

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  Figure 5.. Simulating stretch-dependent changes in Ca2+ signalling in heart A, sarcomere length during a static (0 Hz, blue) and cyclical (2 Hz, red) stretch protocol, the instantaneous probability of NOX2 being in the activated state (πXA, see Eqn (2)), X-ROS factor (χ), and the instantaneous change in single RyR2 PO. B, the mean probability of NOX2 being in the activated state (πXA) during a 10 s stretch of varying magnitudes (i.e. 0, 3, 6, 8 and 10%) and frequencies (i.e. 0, 1 and 2 Hz). Note that a 0 Hz stretch indicates a static, ‘stretch-and-hold’ protocol. C and D, number of Ca2+ sparks (cell−1 s−1) during a static (C) and a cyclical (D) stretch protocol.

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  Figure 6.. Modelling captures stretch-dependent Ca2+ spark behaviour in healthy and dystrophic heart A, representative simulations of transverse confocal linescans for WT and MDX VCMs with an 8% stretch imposed at the red arrow. B, Ca2+ spark frequency, amplitude, FDHM and FWHM before, during and after a static, 8% stretch. Note, simulated transverse linescan frequencies have been adjusted (i.e. divided by 3) to enable direct comparison to experimental longitudinal linescan measures based on an average 600 nm and 1.8 μm inter-CRU distance, respectively. C, simulations of transverse confocal linescans for WT and MDX VCMs exposed to a cyclic, 2 Hz, 10 % stretch. Conditions as follows, WT (WT); MDX (MDX); MDX with 2-fold increase in X-ROS (MDX+); and MDX modified with ‘normalized’ (i.e, equivalent to WT levels) X-ROS components (MDX−). Statistics: MDX compared to WT (*P < 0.05), Stretch compared to Before (#P < 0.05), After compared to Before (§P < 0.05), and After compared to Stretch (‡P < 0.05).

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  Figure 7.. MT densification drives SR Ca2+ leak towards arrhythmogenic levels during cyclic stretch in heart A, simulations of transverse confocal linescans for WT and MDX VCMs exposed to a cyclic, 2 Hz, 10% stretch. Conditions as follows, WT (WT); MDX (MDX); MDX with 2-fold increase in X-ROS (MDX+); and MDX modified with ‘normalized’ (i.e, equivalent to WT levels) X-ROS components (MDX−). B, quantification of RyR release flux. Statistics: Test group (i.e. MDX, MDX+, MDX−) compared to WT (*P < 0.05) within each condition (i.e. Before, Stretch, After), MDX− compared to MDX+ within each condition (§P < 0.05), MDX− compared to MDX within each condition (†P < 0.05), Stretch compared to Before (#P < 0.05), and After compared to Stretch (‡P < 0.05).