Microtubule‐Dependent Ca²⁺ Spark Recruitment in the Heart

In this issue of the Journal of the American Heart Association (JAHA), Fu et al ¹ identified a new class of stretch‐dependent Ca²⁺ signaling events in atrial myocytes of rabbits, pigs, and humans. Such changes in Ca²⁺ signaling could alter force generation or arrhythmogenesis in the affected cells. The team members who did this work were from the Universities of Freiburg, Maastricht, and Ghent, and they showed that diastolic stretch of freshly isolated left atrial cardiomyocytes provoked an increase in the Ca²⁺ spark rate in the stretched region. This increase in the Ca²⁺ spark rate was by a notably different mechanism than the increases in Ca²⁺ spark rate reported by Prosser et al ² in rodent ventricular myocytes because the increase in Ca²⁺ spark rate did not depend on an elevation in local reactive oxygen species (ROS). However, intact microtubules were required for both types of stretch‐induced Ca²⁺ spark increase. Therefore, the term microtubule‐dependent Ca ²⁺ spark recruitment may more accurately describe these findings.

Prosser et al found in 2011 that cellular stretch required intracellular microtubules to activate the NADPH oxidase 2 (NOX2) in the sarcolemmal membrane, and this activation led to an increase in Ca²⁺ sparks in rat ventricular myocytes. ² The activated NOX2 produced extracellular ROS that was thought to reenter the cell via water channels ³ to oxidize nearby intracellular target proteins that led to enhanced Ca²⁺ release from sarcoplasmic reticulum stores. This ROS‐dependent Ca²⁺ signaling mechanism was dubbed “X‐ROS signaling.” Importantly, Fu et al showed that extracellular Ca²⁺ and Na⁺ were necessary for the new class of stretch‐dependent Ca²⁺ signaling events in left atrial myocytes. ¹ They specifically suggested that cation nonselective mechano‐sensitive ion channels were involved. They also provided suggestive evidence that 2 additional specific contributors to the enhanced Ca²⁺ spark activity may be important: (1) the stretch‐activated Piezo1 channel, ⁴ , ⁵ which may have contributed to the process; and (2) that the transient receptor potential ankyrin 1 (TRPA1) channel was central to this signaling. Both Piezo1 and TRPA1 are nonselective cation channels that are permeable to both Na⁺ and Ca²⁺. ² , ⁶ The authors noted that activation of both TRPA1 and Piezo1 channels in left atrial myocytes increased the baseline Ca²⁺ spark rate. However, a further increase in Ca²⁺ spark rate with a subsequent stretch was observed in the presence of the Piezo1 agonist, but not with TRPA1 agonist. See Figure. ¹ , ²

Figure 1. Microtubule‐dependent Ca²⁺ spark recruitment.

A, Graphic comparison of the basic elements in microtubule‐dependent Ca²⁺ spark recruitment by Fu et al ¹ (TRPA1 signaling) and by Prosser et al ² (X‐ROS signaling). B, Microtubule‐dependent Ca²⁺ spark recruitment by TRPA1 signaling involves microtubule‐dependent processes that activate the TRPA1 channels directly and the Piezo1 channels indirectly. C, Microtubule‐dependent Ca²⁺ spark recruitment by X‐ROS signaling when microtubules attached to NOX2 activate ROS production extracellularly that can enter the myocyte via water channels to oxidize proteins nearby. ROS‐dependent changes in the subsarcolemmal proteins may increase the likelihood of Ca²⁺ spark activation. M indicates membrane; NOX2, NADPH oxidase 2; ROS, reactive oxygen species; SL, sarcolemma; TT, transverse tubule; and X‐ROS, NOX2‐ROS.

In support of the novelty of the Fu et al ¹ findings in left atrial myocytes, X‐ROS signaling has been shown to work by very different mechanisms, described by Prosser et al, ² with additional distinctive tissue‐specific components in skeletal and cardiac muscle, ⁷ , ⁸ in metastatic cancer cells ⁹ and in bone osteocytes. ¹⁰ Thus, X‐ROS signaling has many “flavors” and manners of functional expression. Clearly, additional important and exciting atrial investigations will be produced during follow‐up work to this article in left atrial myocytes. A next step in understanding the newly identified microtubule‐dependent Ca²⁺ spark recruitment may depend on more detailed characterizations of the roles of TRPA1 channels in future investigations in atria (Figure). One large area of such investigations may involve the recently reported interactions between NOX2 and TRPA1 channels found in cerebral capillaries reported from the Earley laboratory. ¹¹ Another important area of future work will likely focus on quantitative investigations of Piezo1 biophysics and the responsiveness of these channels to stretch in heart.

Piezo1

Fu et al ¹ showed that the activation of Piezo1 channels alone by Yoda1 could not produce microtubule dependent Ca²⁺ spark recruitment in atrial myocytes. Specifically, Fu et al found that when TRPA1 was first blocked, Piezo1 activation by Yoda1 did not increase the Ca²⁺ spark rate upon stretching. ¹ They thus concluded that (1) TRPA1 may play a direct role in modulating Ca²⁺ sparks via localized Ca²⁺ entry through TRPA1 channel; and (2) Piezo1 and TRPA1 may be functionally coupled. This coupling could be mediated by Ca²⁺, similar to the previously reported interaction between Piezo1 and TRPM4, in which Ca²⁺ influx through Piezo1 activates downstream Ca²⁺‐sensitive TRPA1 channels. While this mechanism appears complex, it is consistent with the previous findings, which suggest that Piezo1 functions as a mechanosensor whereas TRPA1 may not. Likewise, the X‐ROS signaling pathway has been implicated in Piezo1‐mediated mechano‐chemo transduction. Further investigation is needed to assess the functional coupling of Piezo1, TRPA1, and NOX2 across species using knockout models to better define the distinct but microtubule‐dependent signaling pathways involved. Additionally, more quantitative investigations of Piezo1‐dependent Ca²⁺ influx in atrial myocytes are required to determine how Piezo1 contributes to microtubule‐dependent Ca²⁺ spark recruitment involving TRPA1.

TRPA1 Channels

While TRPA1 channels are clearly involved in microtubule‐dependent Ca²⁺ spark recruitment as demonstrated by Fu et al, ¹ the broader literature makes the details of the process potentially complicated. TRPA1 is a nonselective cation channel that permits the influx of Na⁺ and Ca²⁺, thereby modulating intracellular Ca²⁺ homeostasis and downstream signaling pathways. It is expressed in a wide variety of cell types, including but not limited to sensory neurons and endothelial cells (both capillary and arterial). ¹¹ , ¹² , ¹³ In mouse cardiac myocytes, however, the functional expression of TRPA1 has been questioned, ¹⁴ despite the detection of TRPA1 protein using specific antibodies. ¹⁵ Therefore, it is plausible that Fu et al ¹ successfully identified functional TRPA1 channels in rabbit and human atrial myocytes, even though the validation using TRPA1 knockout animal model is required. Indeed, a wide range of TRPA1 expression patterns and subcellular localizations has been reported. Meanwhile, the mechanosensitive properties of TRPA1, as well as those of other mammalian TRP channels, remain a subject of active investigation and debate. While some studies propose that these channels act as primary mechanosensors, ¹⁶ , ¹⁷ others viewed them more as secondary membrane receptors modulated by mechanical stimuli through intermediate signaling cascades. ¹⁸ Supporting its intrinsic mechanosensitivity, single‐channel recordings of reconstituted TRPA1 in artificial lipid bilayers have demonstrated direct mechanosensitive. ¹⁷ Consistent with this, Fu et al confirmed the mechanosensitive behavior of TRPA1 through single‐channel recordings in isolated rabbit atrial myocytes. ¹ Interestingly, despite the clear activation of TRPA1 by the chemical agonist allyl isothiocyanate, evidenced by increased open probability and current amplitude, subsequent mechanical stimulation enhanced channel activity but not Ca²⁺ spark frequency in the presence of allyl isothiocyanate. This observation raises important mechanistic questions about the interplay between chemical and mechanical activation modes of TRPA1. It also aligns with accumulating evidence that oxidative stress modulates TRPA1's response to mechanical stimuli, potentially shifting its gating properties from force‐sensitive to lipid‐sensitive states. This is supported by studies from the Earley laboratory showing that TRPA1 colocalizes with NOX2 in cerebral artery endothelial cells, where endogenous ROS products appear to fine‐tune its function, suggesting a potential convergence between the X‐ROS pathway and TRPA1 activation mechanisms. ¹¹ Notably, the colocalization of TRPA1 and NOX2 was observed in cerebral artery endothelial cells, but such interactions are not observed in other vascular beds, including coronary vessels. These findings indicate that TRPA1's localization and functional integration are subject to regional and cellular specificity, which may have important implications for its role in cardiac physiology and pathophysiology.

Future Work

The magnitude of stretch‐dependent signaling effects will depend on the relative abundance of the sarcolemmal channels as well as the abundance of the relevant and linked microtubules. Transverse tubules in rodent atrial cells appear to be more abundant and complex compared to those in large mammals, and their structure can further change with disease or during development. ¹⁹ , ²⁰ As a result, the membrane surface area in small rodent (i.e mouse) atrial cells is greater than that in large mammals. Thus, the organization of transverse tubules is an important factor to consider when investigating microtubule‐dependent Ca²⁺ spark recruitment. On the other hand, the functional and structural interaction between TRPA1 and microtubules remains largely unexplored. Future studies should also focus on the interplay between microtubule dynamics and the mechano‐chemo transduction, as well as the sensitivity of ryanodine receptors to these regulatory factors.

Disclosures

None.