‘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 (∼300 ms) Ca 2+ transients lasting ∼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 caffeineor 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+ .

CGP-37157 Inhibits the Sarcoplasmic Reticulum Ca2+ATPase and Activates Ryanodine Receptor Channels in Striated Muscle

“…In summary, the literature is replete with studies suggesting that a mitochondrial NCX block by CGP results in intracellular Ca 2ϩ store depletion in various cell systems (Griffiths et al, 1997;Malli et al, 2005;Belmonte and Morad, 2008). In this study, the nature of CGP interactions with SR transporters was explored, and we found that there is direct CGP action on SERCA and RyRs.…”

“…Among mitochondrial transporters, the 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)]-sensitive Na ϩ / Ca 2ϩ exchanger (NCX) may mediate SR Ca 2ϩ load and release (Szalai et al, 2000;Malli et al, 2005;Csordá s and Hajnóczky, 2009). Indeed, CGP has been found to significantly affect intracellular Ca 2ϩ signaling in smooth, cardiac, and skeletal muscle cells and in nonmuscle systems (Griffiths et al, 1997;Malli et al, 2005;Belmonte and Morad, 2008;Liu and O'Rourke, 2008;Chalmers and McCarron, 2009;Csordá s and Hajnóczky, 2009).…”

Effects of hydraulic pressure on cardiomyoblasts in a microfluidic device

“…Belmonte and Morad used pressurized flows (PFs) of solutions to imitate the hemodynamic stress to cardiomyocyte. 10 However, their PFs produced a pressure head of 20.5 mm Hg, which was significantly lower than the blood pressure in human hypertension (higher than 140 mm Hg). Recently, Giridharan et al developed a microfluidic cardiac cell culture model (lCCCM) that incorporated pulsatile stretch and pressure on cardiac cells in a single device.…”

Ca2+ Signaling Triggered by Shear-Autocrine P2X Receptor Pathway in Rat Atrial Myocytes

“…Shear stress of comparable strength induces a distinct spontaneous Ca 2+ transient (timeto-peak, T p : 130 ms) in fura-2-AM-loaded rat atrial myocytes, as shown by epifluorescence measurement [10]. This shear-induced global Ca 2+ signal occurs faster than longitudinal Ca 2+ waves in terms of Ca 2+ release (T p ≥300 ms).…”

“…A series of recent reports have suggested that cardiac myocytes significantly alter their membrane conductance and Ca 2+ signaling in response to shear stress [9][10][11][12][13][14]. It has been reported that shear stress in atrial myocytes enhances Ca 2+ spark occurrence and induces longitudinal Ca 2+ waves under resting conditions [9,12].…”