'Pressure-flow'-triggered intracellular Ca2+ transients in rat cardiac myocytes: possible mechanisms and role of mitochondria

Abstract

Cardiac myocytes, in the intact heart, are exposed to shear/fluid forces during each cardiac cycle. Here we describe a novel Ca(2+) signalling pathway, generated by 'pressurized flows' (PFs) of solutions, resulting in the activation of slowly developing ( approximately 300 ms) Ca(2+) transients lasting approximately 1700 ms at room temperature. Though subsequent PFs (applied some 10-30 s later) produced much smaller or undetectable responses, such transients could be reactivated following caffeine- or KCl-induced Ca(2+) releases, suggesting that a small, but replenishable, Ca(2+) pool serves as the source for their activation. PF-triggered Ca(2+) transients could be activated in Ca(2+)-free solutions or in solutions that block voltage-gated Ca(2+) channels, stretch-activated channels (SACs), or the Na(+)-Ca(2+) exchanger (NCX), using Cd(2+), Gd(3+), or Ni(2+), respectively. PF-triggered Ca(2+) transients were significantly smaller in quiescent than in electrically paced myocytes. Paced Ca(2+) transients activated at the peak of PF-triggered Ca(2+) transients were not significantly smaller than those produced normally, suggesting functionally separate Ca(2+) pools for paced and PF-triggered transients. Suppression of nitric oxide (NO) or IP(3) signalling pathways did not alter the PF-triggered Ca(2+) transients. On the other hand, mitochondrial metabolic uncoupler FCCP, in the presence of oligomycin (to prevent ATP depletion), reversibly suppressed PF-triggered Ca(2+) transients, as did the mitochondrial Ca(2+) uniporter (mCU) blocker, Ru360. Reducing agent DTT and reactive oxygen species (ROS) scavenger tempol, as well as mitochondrial NCX (mNCX) blocker CGP-37157, inhibited PF-triggered Ca(2+) transients. In rhod-2 AM-loaded and permeabilized cells, confocal imaging of mitochondrial Ca(2+) showed a transient increase in Ca(2+) on caffeine exposure and a decrease in mitochondrial Ca(2+) on application of PF pulses of solution. These signals were strongly suppressed by either Na(+)-free or CGP-37157-containing solutions, implicating mNCX in mediating the Ca(2+) release process. We conclude that subjecting rat cardiac myocytes to pressurized flow pulses of solutions triggers the release of Ca(2+) from a store that appears to access mitochondrial Ca(2+).

Figure 1. Measurement of Ca²⁺ transients in fura-2 AM-loaded atrial myocytes

A, characteristic activation sequence for ‘pressurized flow’ (PF)-triggered Ca²⁺ transients in atrial myocytes incubated in 5 mm Ca²⁺-Tyrode solution: 1 Hz electrical stimulation (indicated by *), 2 s application of 10 mm caffeine and washout, and PF of solution for 2 s in consecutive episodes. Note that caffeine washout was accomplished by switching the ‘puffing’ solution from caffeine-containing Tyrode to control Tyrode. B, summarized data from A comparing the magnitude of the indicated Ca²⁺ transients. C, histogram comparing the magnitude of the ratio of PF to paced Ca²⁺ transients in the same cell. D, accumulated data with rise kinetics for the three types of Ca²⁺ transients. *P < 0.001 versus PF. E, alternative protocol for PF-triggered Ca²⁺ transient activation utilizing 75 mm KCl to raise [Ca²⁺]_i. F, summarized data from E comparing the magnitude of the indicated Ca²⁺ transients.

Figure 2. Comparison of Ca²⁺ transients in fura-2 AM-loaded atrial and ventricular myocytes

A, atrial (upper traces) and ventricular myocytes (lower traces) were incubated in 5 mm Ca²⁺-Tyrode (bath) solution and electrically stimulated at 1 Hz (*). Next, myocytes were subjected to brief (2 s) pulses of 10 mm caffeine and PFs, according to the activation sequence previously described. B, summarized atrial data for the three indicated Ca²⁺ transients. *P < 0.001 versus pace. C, summarized ventricular data for the three indicated Ca²⁺ transients.

Figure 3. L-type Ca²⁺ channel, NCX, or SAC blockade does not suppress PF-triggered Ca²⁺ transients in atrial myocytes

A, control PF-triggered Ca²⁺ transients were activated (upper traces) before 1 mm Cd²⁺ was applied in the caffeine washout and PF solution (lower traces). B, summary of data from A comparing the magnitude of PF-triggered Ca²⁺ transients under control and Cd²⁺ conditions. C, Ca²⁺ transients were first activated by PF of Ca²⁺-free solution (upper trace). Ni²⁺ at 5 mm was then used in the caffeine washout and PF solution to activate subsequent PF-triggered Ca²⁺ transients (lower traces). D, summary of data from C comparing the magnitude of the indicated PF-triggered Ca²⁺ transients. *P < 0.05 versus Ca²⁺ free. E, control PF-triggered Ca²⁺ transients were activated (upper traces) before 100 μm Gd³⁺ was used in both caffeine washout and PF solution to generate subsequent PF-triggered Ca²⁺ transients (lower traces). F, summary of data from E comparing the magnitude of PF-triggered Ca²⁺ transients under control and Gd³⁺ conditions.

Figure 4. PF-triggered Ca²⁺ release in paced and resting atrial myocytes

A, cells were electrically stimulated at 0.75 Hz for 2 min before PF-triggered Ca²⁺ transients were activated. B, after electrical stimulations had been stopped for 2 min, PF-triggered Ca²⁺ transients were markedly reduced. C, the magnitude of PF-triggered Ca²⁺ transients returned to pre-quiescent levels following resumption of electrical stimulation for 2 min. Traces are representative of experiments performed on 8 atrial myocytes.

Figure 5. Interaction of PF-triggered and electrically activated Ca²⁺ transients

A, atrial myocytes were electrically stimulated (*) before a 1 s caffeine pulse was applied. Washing away caffeine (a) at the peak of caffeine-induced Ca²⁺ release produced a small PF-triggered Ca²⁺ transient. Electrically stimulated Ca²⁺ transients (c) could be activated at the peak of PF-triggered Ca²⁺ transients (b), without affecting subsequent paced Ca²⁺ transients (d). Traces are representative of experiments performed on 10 cells. B and C, magnification of traces from A. D, when caffeine is not washed away by a jet of washout solution, [Ca²⁺]_c declines very slowly and no PF-triggered Ca²⁺ transient is apparent.

Figure 6. IP₃R blocker 2-APB and NOS inhibitor L-NAME do not inhibit PF-triggered Ca²⁺ transients

A, PF-triggered Ca²⁺ transients were activated in atrial myocytes in control conditions (upper traces) and after perfusing the bath solution with 1 μm 2-APB for 9 min (lower traces). B, summarized data from A showing mean magnitude of PF-triggered Ca²⁺ transients. C, PF-triggered Ca²⁺ transients were activated in atrial myocytes in control conditions (upper traces) and after perfusion of 1 mm l-NAME in the bath solution for 10 min (lower traces). D, summarized data from C showing mean magnitude of caffeine-induced and PF-triggered Ca²⁺ transients.

Figure 7. Mitochondrial uncoupler FCCP inhibits PF-triggered Ca²⁺ transients

A, PF-triggered Ca²⁺ transients were activated in control conditions (upper traces), and compared to PF-triggered responses generated after 0.5 μm FCCP and 5 μm oligomycin were used in the caffeine washout solution for 30 s (middle traces). PF-triggered Ca²⁺ transients returned to control levels after a 2 min FCCP washout period (lower traces). B, summarized data from A showing mean magnitude of the indicated PF-triggered Ca²⁺ transients. *P < 0.05 versus control PF pulse.

Figure 8. Mitochondrial Ca²⁺ uniporter blocker Ru360 significantly reduces PF-triggered Ca²⁺ transients

A, PF-triggered Ca²⁺ transients were activated under control conditions (upper traces) and after treatment of atrial myocytes with 5 μm Ru360 for 10 min (lower traces). B, summarized data from A showing mean magnitude of various Ca²⁺ transients. *P < 0.001 versus control PF pulse.

Figure 9. ROS scavenger, tempol, and disulphide-reducing agent, dithiothreitol (DTT), inhibit PF-triggered Ca²⁺ transients

A, PF-triggered Ca²⁺ transients were activated in atrial myocytes under control conditions (upper traces) and after perfusing 100 μm tempol in the bathing solution for 2 min (middle traces). The tempol effect was reversible after a 3–5 min washout period (lower traces). B, summary of data from A showing the mean magnitude of paced and PF-triggered Ca²⁺ transients under the condition indicated. *P < 0.05 versus control PF. C, PF-triggered Ca²⁺ transients were activated in atrial myocytes under control conditions (upper traces) and after perfusing 2 mm DTT in the bathing solution for 4 min (middle traces). The DTT effect was reversible after 4–5 min of DTT washout (lower traces). D, summary of data from C showing the mean magnitude of paced and PF-triggered Ca²⁺ transients under the condition indicated. *P < 0.05 versus control PF.

Figure 10. Mitochondrial Ca²⁺ measurements using confocal microscopy

A, [Ca²⁺]_m was measured in permeabilized ventricular myocytes loaded with 10 μm rhod-2 AM for 60 min at room temperature. Myocytes were exposed to caffeine, PF and CCCP, and imaged confocally at either 7.5 Hz (upper and middle traces) or 0.3 Hz (lower trace). The shutter was closed between image acquisitions to prevent photobleaching when UV light exposure exceeded 30 s. B, representative images of a myocyte before (1) and at the peak (2) of the indicated treatment, corresponding to the points indicated in A. Fluorescence colour scale (0–22) is common to all images. C, summary of data from A showing the mean peak fluorescence change for the indicated treatments. Fluorescence was normalized to the baseline fluorescence (F₀) before the various treatments.

Figure 11. Comparison of the kinetics of Ca²⁺ signals in intact and permeabilized ventricular myocytes

A, 7 representative traces from intact fura-2 AM-loaded cells were normalized and superimposed on each other to demonstrate the time course of global cytosolic Ca²⁺ changes on caffeine (left traces) or PF (right traces) application. B, 7 representative traces from permeabilized myocytes incubated with rhod-2 AM were normalized and superimposed on each other, showing the time course of mitochondrial Ca²⁺ changes on caffeine (left traces) or PF (right traces) application. The traces in A and B have identical x-axis scales, allowing easier visual comparison of the time course of events. C, summary of data. *P < 0.01 versus caffeine in intact myocyte; ΨP < 0.01 versus PF in intact myocyte.

Figure 12. PF-induced mitochondrial Ca²⁺ signals are completely suppressed in permeabilized ventricular myocytes perfused with Na⁺-free solution

A, [Ca²⁺]_m was measured in permeabilized ventricular myocytes loaded with 10 μm rhod-2 AM for 60 min at room temperature. Myocytes were perfused with internal bathing solution containing 10 mm Na⁺ and subjected to a control caffeine pulse. The insets are expanded images from the section of the cell indicated in Aa, and corresponding to the points 1 and 2 on the trace. Aa, confocal image of a representative myocyte, filtered as described in Methods. Fluorescence colour scale (0–22) is common to all images. B, subsequent to caffeine treatment, a control PF response was activated. C, after 5 min in Na⁺-free solution (Na⁺ replaced in equimolar amounts by K⁺), caffeine in Na⁺-free solution was applied. D, PF pulse of Na⁺-free solution was given after caffeine treatment.