Diastolic stretch gives rise to an acute and transient increase in Ca2± spark rate, without changing the dynamic properties of individual sparks. Using the whole-cell stretch protocol (which has the advantage of maximizing the observation area), it was established that the stretch-induced increase in Ca2± spark rate (to 130.7±6.4% of control) occurs acutely, within the first 5 seconds (minimum observation period with sufficient power to conduct statistical analysis), and that this increase is of a transient nature, with spark rate returning to near control levels (104.4±5.1%) within 1 minute. This response cannot be explained by spatial rearrangement of RyR2, even though constant volume behavior predicts radial compression (by approximately −4%) during axial strain (by ±8%). Because the total number of RyR2 in the stretched cell portion does not change either, net density of RyR2 in any subvolume that is large compared to the dimensions of a single sarcomere is unlikely to show a significant change. In addition, the transient nature of the observed spark rate response (in the presence of maintained stretch) makes it unlikely that spatial rearrangement of RyR2 plays a significant role in this context.
A possible explanation for the initial increase in spark rate would be that axial cell stretch causes membrane depolarization, promoting Ca2± influx that could stimulate sparks. However, the local nature of the stretch-induced increase in Ca2± spark rate, established in half-cell stretch experiments ( and ), argues against any mechanism of inherently whole-cell nature.
Alternatively, stretch could increase transsarcolemmal Ca2± influx, perhaps via SAC, to an extent that might be small enough to have no effect on the membrane potential, while still acting locally to promote SR Ca2± release events. However, the lack of an effect of either GsMTx-4 exposure () or perfusion with 0NC solution () suggests that any contribution of trans-sarcolemmal Ca2± fluxes to the acute stretch-induced increase in Ca2± spark rate must be negligible.
This may be different during sustained axial stretch where, as previously illustrated by Gannier et al, an increase in resting [Ca2±
may occur via a streptomycin-sensitive mechanism (streptomycin also blocks SAC24
), perhaps involving Ca2±
influx via SAC, or secondary effects of Na±
influx via SAC on the Na±
This may contribute to an involvement of SAC in the slow force response to stretch (which is accompanied by an increase in [Ca2±
However, these mechanisms appear to require time periods of 5 minutes or more to affect cell function. In contrast, the acute increase in Ca2±
spark rate observed here occurs in resting cells, within the first 5 seconds of stretch application, and even in the absence of extracellular Ca2
Similar time constraints appear to apply to NO-mediated effects of stretch on Ca2
spark rate, which have been reported on exposure of rat cardiomyocytes to 10 minutes stretch.12
The acute stretch-induced increase in Ca2
spark rate observed here is of a transient nature () and involves different mechanisms, as it is not blocked by preincubation with L-NAME (). In common with the previously reported late effects of stretch on Ca2
our data show no changes in spark amplitude, time to peak, or decay time constant (). This suggests that stretch is unlikely to act via an increase in the Ca2
conductance of individual RyR2 or in the number of RyR2 recruited in a spark event cluster.
An alternative explanation is that axial cell stretch, via a hitherto unidentified pathway, increases the open-probability of RyR2. This could cause an acute increase in Ca2
spark rate, enhancing SR Ca2
leak, and thereby causing partial depletion of the SR (as Ca2
, released into the dyadic cleft, will only partially be pumped back into SR, and partially extruded from the cell via Na/Ca2
exchanger). Such early stretch-induced reduction in [Ca2]SR
has been reported before.8,9
is an important driver of Ca2
this would allow the cell to return to near-equilibrium Ca2
spark rates, once the opposing effects of stretch and SR Ca2
load on RyR2 open probability have balanced out.
Several studies have discussed an involvement of the cytoskeleton in Ca2
handling, partially with contradictory results.22,23,29
Most recently, cardiomyocytes from the murine model of Duchene’s muscular dystrophy (ie, the dystrophin null MDX mouse) have been reported to respond to increased mechanical loads (whether applied as axial stretch by CFs30
or via osmotic swelling31
) with an augmentation in SR Ca2
release. Of particular interest, in this context, is the observation that among the compensatory adaptations in the MDX heart there is an 1.4-fold increase in/3-tubulin,32
which, based on our findings, may strongly contribute to mechanically-promoted SR Ca2
release in this disease model.
Our findings highlight that microtubule integrity is obligatory for the acute stretch-induced increase in Ca2+
spark rate (). The actual mechanisms underlying this involvement of the cytoskeleton are not clear. Based on the close proximity of microtubules with SR and T-tubular membranes (10−8
m; ), one might speculate on the possibility of physical transmission of stress or strain from sarcolemmal CF attachment points to RyR2 or membrane areas near RyR2 via microtubules. As major force-bearing components of the nonsarcomeric cytoskeleton, microtubules contribute to cardiomyocyte stiffness during axial compression (when microtubules “buckle,” contributing to passive load and cell recoil), although they appear not to affect tensile or viscoelastic behavior during axial elongation (which is best explained by their ability to translocate in the direction of positive strain).33–36
Microtubules are laterally enforced, both by the cytosolic viscosity and by direct elastic cytoskeletal links, as can be illustrated by the observation that neighboring microtubules in cardiomyocytes “often buckle … in a coordinated manner, both temporally and spatially in phase.”37
Such coordinated buckling of microtubules in cardiomyocytes has been observed over distances in the 10−6
m region, highlighting the plausibility of mechanical interference between microtubules and the T-tubular–SR membrane complex.
It is possible, therefore, that microtubules mechanically interfere with the SR in a way that may affect RyR2 open probability in a manner akin to SAC activation. The approximately 102
RyR2 receptors within the Ca2+
release unit are thought to interact with each other mechanically, contributing to the coordinated generation and termination of Ca2+
and it is only a modest extension of this notion to suggest that RyR2 gating may be mechanically influenced by microtubule-mediated perturbation of the T-tubular–SR membrane complex. Similar deformation-induced increases in Ca2+
spark rate have been observed in the depth of atrial myocytes during sarcolemmal fluid-jet stimulation.39
Also, RyR2 mechanosensitivity could underlie the fluid-pressure induced increase in Ca2+
releasability from the SR, observed in rat ventricular cardiomyocytes.40
Alternatively, there may be mitochondria-mediated responses,39
or currently unknown effects of fast-acting local signal transduction pathways that are relevant for RyR2 function and affected by the cytoskeleton.41
In conclusion, axial stretch of rat cardiomyocytes acutely and transiently increases Ca2+ spark rate via pathways that are independent of SAC, NO, and transsarcolemmal Ca2+ influx but that do require cytoskeletal integrity. The mechanisms, interplay, and functional relevance of acute and late stretch effects on Ca2+ spark rate, as well as the interrelation of cytoskeletal elements withCa2+ handling cell structures, form worthwhile targets for further elucidation.