Following an acute injury to the heart, as in myocardial infarction, fibroblasts participate in the wound healing process. Because of the limited capability of cardiomyocytes to regenerate, wound healing concludes with loss of ventricular muscle and formation of a stable scar, ultimately leading to a fibrotic, arrhythmogenic substrate.9
The myofibroblast repairs the injured region through the formation of granulation tissue,5,18
using contraction to reduce the total scar volume.19
It is well known that mechanoelectric coupling among cardiac cells, the process in which mechanical perturbations alter electrical activity, exists in the intact heart20
and in cardiac cell cultures.21
Cardiomyocytes also possess MSCs whose open probability increases with stretch22
and whose mechanical sensitivity increases following myocardial infarction.23
Thus, we tested the hypothesis that increased vulnerability to arrhythmias following cardiac injury could be related to a tonic tugging force that myofibroblasts exert on the cardiomyocyte membrane through mechanical coupling and, lead to conduction slowing and block through the action of MSCs.24
We used TGF-β in our fibrotic models to induce fibroblast differentiation to contractile myofibroblasts. Although fibroblasts in culture transition to proto-myofibroblasts that express some stress fibers,18
incubation with TGF-β increases complete differentiation to myofibroblasts,25
expression of SMA,5
and generation of strong contractile forces (). Our results demonstrate that co-cultures of cardiomyocytes and untreated fibroblasts have a significant reduction in conduction velocity compared with NRVC control monolayers, similar to previous reports in the literature for co-cultures of cardiomyocytes with non-TGF-β treated fibroblasts.11
However, our experiments involving the supplementation of NRVC monolayers with TGF-β treated myofibroblasts suggest that conduction slowing is exacerbated well beyond that obtained with supplementation using untreated fibroblasts (), without a concordant increase in Cx43 expression ().
The recent perspective that fibroblasts may have an active electrical influence on cardiac electrophysiology has been bolstered by discoveries of in vitro
and in situ
gap junctional coupling between cardiac fibroblasts and cardiomyocytes,3,26
and by characterization of multiple fibroblast ion channels,27
including K channels,28
and mechanosensitive channels.29
Numerous studies have focused on the potential electrophysiological consequences of electrical coupling between fibroblasts and cardiomyocytes,30–32
and the general dogma of these studies is that through electrical coupling, fibroblasts actively depolarize resting cardiomyocytes because of their more positive resting membrane potential.
However, in our studies, immunolabeling for Cx43 and pan-cadherin of fibroblast-only cultures suggests a different mechanism by which myofibroblasts and cardiomyocytes can interact. We found that SMA-negative fibroblasts expressed abundant Cx43, whereas SMA-positive myofibroblasts had little Cx43 expression but enhanced pan-cadherin expression (), suggesting that myofibroblasts form strong mechanical rather than electrical junctions. However, TGF-β treated myofibroblasts did have significantly more soluble Cx43 (), indicating that Cx43 is still present in the cytoplasm. This finding is consistent with recent evidence demonstrating that Cx43 is necessary for fibroblast differentiation into myofibroblasts.33
In heterocellular contacts between adjoining cardiomyocytes and myofibroblasts, we also found that adherens junction expression dominated over gap junction expression (). Our key finding that myofibroblast-induced slowing of conduction could be restored with contraction or MSC blockers (, ) was unchanged with connexin43 silencing in the myofibroblasts (). Further, given that changes in myofibroblast contractile force () paralleled the changes in conduction velocity brought about by both TGF-β and blebbistatin (,,), and considering the plethora of work demonstrating the significance of myofibroblast contraction in other tissues,5
we believe that mechanical coupling between myofibroblasts and cardiomyocytes is an important factor contributing to conduction slowing in co-cultures of these two cell types. We suggest that this form of mechanical signaling can activate MSCs that depolarize the cardiomyocyte membrane,34
thereby inactivating the Na+
channels that drive conduction. Further, heterogeneity in the distribution of myofibroblasts can accentuate spatial gradients in conduction and action potential duration, leading to increased propensity to reentrant arrhythmias that again, may be suppressed by MSC or contraction blockers (). As a caveat, we cannot rule out the possibility of paracrine signaling between myofibroblasts and cardiomyocytes as a means of modulating cardiac conduction, although the continuous exchange of solution in our experimental setup suggests that paracrine factors do not play a dominant role.
We believe that our in vitro
model, consisting of longitudinally patterned myocytes and adjacent contractile SMA myofibroblasts, retains several key aspects of the environment in a healing infarct. Previous studies have shown that both SMA positive myofibroblasts35
and TGF-β are markedly expressed36
during infarction, and can remain elevated for several months to years in the infarct border zone.37,38
Further, although numerous studies have shown that Cx43 is downregulated and redistributed post infarction,39,40
cadherin expression levels and distribution remain unaffected or are downregulated to a lesser degree compared with Cx43.40
On the other hand, our results are specific to in vitro
conditions, and it remains to be seen whether they extrapolate to the in vivo
heart in light of known differences between the two environments. First, our cells are maintained under culture conditions (culture media, 2D rigid substrate, lack of hemodynamic loading), which can affect cell shape, protein expression and cell function.21
Second, the spatial distribution of heterocellular gap junctions and adherens junctions in cell monolayers may differ significantly from that found in vivo
. Although myofibroblast-myocyte junctions have not been characterized in the intact heart, in normal myocardium, myocyte-myocyte junctions occur primarily within intercalated disks, in contrast to their distribution around the cell perimeter in cell culture,21
although in infarcted myocardium the distribution also tends to occur around the cell perimeter.40
If future studies confirm our findings in infarcted regions of the heart in vivo
, it is plausible that mechanosensitive channel blockers or fibroblast-specific contraction inhibitors may provide a means to reduce the incidence of arrhythmias.
To conclude, our data demonstrate that impaired conduction in an in vitro fibrotic model is mechanically dependent. Furthermore, our findings support the current view that myofibroblasts are capable of actively decreasing conduction among cardiomyocytes, and suggest that mechanical coupling between myofibroblasts and cardiomyocytes can play a more prominent role in this regard than electrical coupling. Finally, we propose a novel mechanism in which myofibroblasts may impair cardiomyocyte electrophysiological function through the application of contractile force to the cardiomyocyte membrane and activation of mechanosensitive channels.