Microtubule-Dependent Mitochondria Alignment Regulates Calcium Release in Response to Nanomechanical Stimulus in Heart Myocytes

Abstract

Arrhythmogenesis during heart failure is a major clinical problem. Regional electrical gradients produce arrhythmias, and cellular ionic transmembrane gradients are its originators. We investigated whether the nanoscale mechanosensitive properties of cardiomyocytes from failing hearts have a bearing upon the initiation of abnormal electrical activity. Hydrojets through a nanopipette indent specific locations on the sarcolemma and initiate intracellular calcium release in both healthy and heart failure cardiomyocytes, as well as in human failing cardiomyocytes. In healthy cells, calcium is locally confined, whereas in failing cardiomyocytes, calcium propagates. Heart failure progressively stiffens the membrane and displaces sub-sarcolemmal mitochondria. Colchicine in healthy cells mimics the failing condition by stiffening the cells, disrupting microtubules, shifting mitochondria, and causing calcium release. Uncoupling the mitochondrial proton gradient abolished calcium initiation in both failing and colchicine-treated cells. We propose the disruption of microtubule-dependent mitochondrial mechanosensor microdomains as a mechanism for abnormal calcium release in failing heart.

Graphical abstract

Figure 1

Schematic Representation of the Experimental Protocol (A) Cells were loaded with 5 μmol/l of Fluo-4 AM, and a 10 × 10 μm of cell surface was scanned with the SICM. The nanopipette was positioned above a crest or a groove as identified on the scan and, while keeping the distance constant at 200 nm, positive air pressure was applied to the auxiliary port of the pipette holder, generating a hydrojet pressure. A protocol written on Clampfit 10.0 (Molecular Devices) synchronized the light shutter for optical acquisition (8 s in total at 1–5 KHz temporal acquisition) and the pump for pressure application (ramp duration 2 s at 20 kPa). Fluo-4 fluorescence emission was recorded (represented as a color-coded time-lapse map) together with the Z-piezo displacement (corresponding to membrane indentation) and the mechanically induced calcium initiation and propagation. Typical readings of pressure, Z-piezo displacement, and calcium transient are represented on the right. (B) Perturbed area following 20 kPa ramp hydrojet pressure (2 s) in an isolated cardiomyocyte. Pipette solution was filled with 1 μM Lucifer yellow, resulting in ∼0.125 μm² area (green spot), enlarged in the inset. The scale bar represents 10 μm in (A) and 500 nm in the inset.

Figure 2

MiCa_i Propagation Changes from Local to General during Progression to Heart Failure (A) (Upper left) Surface topography of an AMC cardiomyocyte (10 × 10 μm). (Upper right) Surface topography of a heart failure cardiomyocyte (10 × 10 μm) at 4 weeks post-myocardial infarction (MI_4wks) is shown. (Lower left) MI_8wks surface topography is shown. (Lower right) MI_16wks surface topography is shown. (B) Frequency of MiCai propagation during progression to heart failure at AMC, 4–8, and 16 weeks post-MI, respectively. (C) Z-groove index calculated for AMC cells and heart failure cells at 4, 8, and 16 weeks post-MI (n = 6 each; mean ± SEM; ^∗p < 0.0005). (D) Membrane compliance calculated after 20 kPa hydrojet square pulse pressure applied for 2 s at crests, Z-grooves, or un-striated parts of the cells. Pipette-tip diameter 200 nm; n: approximately 20 cells each group; n = 71 in total; mean ± SEM; ^∗p < 0.05; ^∗∗p < 0.001.

Figure 3

MiCa_i Propagation Changes from Local to General during Progression to Heart Failure (A) Color-coded propagation time maps of MiCa_i in an AMC (focal propagation; left panel) and a failing cardiomyocyte 16 weeks post-MI (whole-cell propagation; right panel). The scale bar represents 10 μm. (B) Mechanically induced calcium transient (MiCa_i) parameters (time-to-peak, duration, and amplitude; mean ± SEM) in AMC cells when the pressure was applied either to a crest or to a groove and in heart failure cells to unstructured areas; n = 10 each. (C) Frequency of MiCa_i in AMC cells at baseline (upper left), in the presence of caffeine (upper right), nifedipine (lower left), CCCP (lower middle), and CsA (lower right); n = 14. n.s., not significant; p = 0.0005 (MI-16wks); Fisher exact test; multiple contingency.

Figure 4

Myocardial-Infarction-Induced Remodeling of Dyad Microdomains Is Characterized by a Mitochondrial Shift (Left column) Control (AMC) cells; (right column) heart-failure-derived cells (16 weeks post-MI). (Top row) SICM surface topography is shown; (next row down) TMRM-labeled mitochondria are shown; (next row down) merged images of SICM cell topography and surface confocal (10 × 10 μm) are shown; and (bottom row) representative transmission electron micrographs, illustrating the reorganization of mitochondria in heart failure, are shown.

Figure 5

Disruption of Microtubules Leads to a More-Frequent MiCa_i (A) Time-lapse color-coded maps of MiCa_i. (Top row) 20 kPa hydrojet pressure applied to the center of an AMC myocyte produces no MiCa_i; (bottom row) the same cell after exposure to 10 μmol/l colchicine for 1 hr at 36°C shows a propagated MiCa_i after the same pressure has been applied to the same spot. The scale bar represents 10 μm. (B) Membrane staining of T-tubules (green; Di-8-ANNEPS) and immunostaining for β-tubulin (red) in an AMC cardiomyocyte (left panels) and an AMC cardiomyocyte incubated with colchicine for 1 hr in 36°C (right panels). The scale bar represents 10 μm. (Rightmost picture) Electron micrograph shows mitochondrial movement following incubation of an AMC cell with colchicine (10 μmol/l for 1 hr). The scale bar represents 1 μm. Mit, mitochondria. (C) Frequency of propagated MiCa_i that occur in AMC cells treated with colchicine and with colchicine in combination with CCCP. n = 12 AMC; n = 21 colchicine; n = 10 colchicine+CCCP. p = 0.0005; Fisher exact test; multiple contingency. (D) Membrane compliance of crests and grooves in AMC treated with colchicine and CCCP. n = 21 per group; ^∗∗p < 0.001.

Figure 6

MiCa_i Occurrence in Human DCM Cardiomyocytes (A) Membrane topography of a human heart failure cardiomyocyte (10 × 10 μm). (B) (Left-hand side) color-coded time-lapse map of MiCa_i propagation. The scale bar represents 10 μm. (C) Fluorescence trace of MiCa_i. (D) Frequency of propagated MiCa_i in human heart failure cells. (E) Microtubule protein mRNA is upregulated in DCM cardiomyocytes as compared to non-failing human cardiomyocyte. Technical triplicate normalized to 18 s is shown. mRNA quantities are presented as mean ± SEM (^∗∗p < 0.01); n = 7.

Figure 7

Schematic Representation of the Proposed Mechanisms of MiCa_i Propagation (A) Normal conditions. The interplay of an organized microtubular network, regular T-tubule membrane structure, and sub-sarcolemmal mitochondrial alignment protects against MiCa_i propagation by providing tight control of calcium levels. MIT, mitochondria; SR, sarcoplasmic reticulum. (B) Heart failure conditions. Overexpression and remodeling of microtubules together with mitochondrial delocalization and loss-of-membrane structural regularity enable MiCa_i propagation due to loss of appropriate control.