Pathological left ventricular hypertrophy (LVH) is the most powerful independent predictor for cardiovascular mortality (Levy et al., 1990; Mudd and Kass, 2008). It occurs in response to two very common clinical conditions: systemic hypertension and aortic valve stenosis. It manifests as increased cardiomyocyte volume and weight (Frey et al., 2004; Nicks et al., 2020; Swynghedauw, 1999; Wilde and Brugada, 2011), which results in increased heart mass, particularly left ventricular (LV) mass. Although pathological LVH commonly occurs as a response to increased cardiac wall stress, sometimes termed ‘compensatory hypertrophy’, it is now well established that the effects of pathological LVH are deleterious for heart function, leading to increased cardiac failure and death (Levy et al., 1990; Mudd and Kass, 2008). So far, the only treatment for this condition is lowering elevated blood pressure or replacing a stenotic aortic valve. However, these treatments cannot completely reverse the pathological effects on the myocardium once LVH is established. Consequently, understanding the molecular mechanisms underlying pathological LVH may lead to therapies directed at preventing, inhibiting, or reversing pathological LVH and reducing its associated morbidity and mortality.

The development of pathological LVH depends on upstream stimuli, such as mechanical forces (e.g. pressure overload) or neuroendocrine hormones (e.g. angiotensin II), and distinct downstream signalling mechanisms (Maillet et al., 2013; Nakamura and Sadoshima, 2018; Saucerman et al., 2019; Tham et al., 2015; van Berlo et al., 2013). Importantly, a large body of work implicates intracellular Ca²⁺ levels and subsequent activation of Ca²⁺/calmodulin (CaM)-dependent signalling pathways, such as the calcineurin-nuclear factor of activated T-cells (NFAT)-GATA4 axis, in the induction of pathological LVH (Bers, 2008; Kehat and Molkentin, 2010; Molkentin, 2000; Molkentin, 2013; Molkentin et al., 1998; Wilkins et al., 2004; Wilkins and Molkentin, 2004; Zarain-Herzberg et al., 2011). Gq-coupled receptors are thought to play an important role in the induction of pathological LVH in response to both neurohumoral stimulation (Adams et al., 1998; Nakamura and Sadoshima, 2018; Paradis et al., 2000) and mechanical forces, such as the increase in LV afterload induced by experimental aortic constriction (Keys et al., 2002). Once activated, Gq-coupled receptors are thought to then activate the calcineurin-NFAT pathway (Kehat and Molkentin, 2010; Molkentin, 2000; Molkentin, 2013; Molkentin et al., 1998; Wilkins et al., 2004; Wilkins and Molkentin, 2004).

Our previous experimental work, however, has demonstrated that although Gq-coupled receptors and the calcineurin-NFAT pathway are essential for the induction of LVH in response to angiotensin II, neither are required for the induction of LVH in response to transverse aortic constriction (TAC), the most common experimental model of LV pressure overload (Yu et al., 2021), and one not associated with activation of the renin–angiotensin system (Zou et al., 2004). In contrast to the lack of activation of the calcineurin-NFAT pathway with TAC, an alternative Ca²⁺/CaM-dependent signalling pathway, the Ca²⁺/CaM-dependent protein kinase II (CaMKII)-histone deacetylase (HDAC)-myocyte enhancer factor 2 (MEF2) pathway (Backs et al., 2009; Backs et al., 2006; Passier et al., 2000), is activated in response to TAC (Yu et al., 2021).

Left unexplained by our previous work, however, is the mechanism by which the CaMKII-HDAC-MEF2 pathway is activated by TAC, given that this activation is not dependent on Gq-coupled receptors. Prime candidates for mediating this mechanism are mechanosensitive ion channels. In cardiac mechanotransduction, where mechanical stimuli are converted into electrical or chemical signals (Martinac and Cox, 2017; Peyronnet et al., 2016), Ca²⁺-dependent ion channels, such as transient receptor potential (TRP) channels, act as important modulators of intracellular Ca²⁺ homeostasis (Wang et al., 2018) and are thought to be unique biosensors that activate specific pathological LVH signalling pathways (Eder and Molkentin, 2011; Wu et al., 2010). As a Ca²⁺- and voltage-activated non-selective monovalent cation channel, TRP cation channel subfamily melastatin 4 (TRPM4) may contribute to an increase in intracellular Ca²⁺ concentration by causing membrane depolarisation (Launay et al., 2002), although we and others (Constantine et al., 2016; Gottlieb et al., 2008; Nikolaev et al., 2019) have demonstrated that mammalian TRP channels, including TRPM4, are not directly activated by membrane stretch. Consequently, if TRPM4 plays a role in TAC-induced LVH, it acts as an amplifier of the primary Ca²⁺ or voltage signal from a yet to be determined mechanosensitive ion channel or channels. TRPM4 has been functionally characterised in atrial and ventricular cardiomyocytes, both human and rodent (Guinamard et al., 2004; Guinamard et al., 2006; Watanabe et al., 2008). Other studies indicate that TRPM4 contributes to both cardiac function and disease development, including cardiac hypertrophy and heart failure (Frede et al., 2020; Gueffier et al., 2017; Hedon et al., 2021; Jacobs et al., 2015; Mathar et al., 2014). Previous studies using Trpm4 cardiomyocyte-specific knock-out (Trpm4 cKO) mice have shown that TRPM4 is a negative regulator of angiotensin II-induced cardiac hypertrophy in mice, which involves the calcineurin-NFAT pathway (Kecskés et al., 2015), and that TRPM4 is essential for survival after myocardial infarction (Hedon et al., 2021; Jacobs et al., 2015). However, whether TRPM4 plays a role in mechanical pressure overload-induced LVH has yet to be determined.

Here, we investigate the role of TRPM4 in pressure overload LVH induced by TAC in homozygous Trpm4 cKO mice (Kecskés et al., 2015) as compared to wild-type (WT) control mice. We demonstrate that loss of cardiomyocyte TRPM4 significantly attenuates the development of LVH observed in response to TAC in WT mice. Moreover, this effect is associated with reduced activation of the CaMKII-HDAC4-MEF2 pathway.

In the present study, we employed mice subjected to TAC as an in vivo cardiac hypertrophy model to investigate the role of the TRPM4 ion channels in pressure overload-induced pathological LVH. We compared Trpm4 cKO mice with WT controls. The experimental animals were examined 2 days after surgery when the molecular signalling pathway that drives LVH is switched on in response to the increased haemodynamic load induced by TAC but, importantly, before LVH has developed. In addition, the experimental animals were examined 14 days after surgery when the TAC-induced LVH phenotype is evident.

First, we found that TRPM4 channel expression in the WT mouse heart was modified by TAC-induced pressure overload hypertrophy. At 2 days and 14 days after TAC, both Trpm4 mRNA and protein expression were downregulated in LV tissue and isolated cardiomyocytes, suggesting that TRPM4 plays a role in TAC-induced LVH. Second, we demonstrated that the role of TRPM4 was pro-hypertrophic by performing sham and TAC surgery in Trpm4 cKO mice. This demonstrated that a reduction in TRPM4 expression in cardiomyocytes dampens the hypertrophic response to TAC, as evident by an approximately 50% reduction in the degree of LVH and LV fibrosis in Trpm4 cKO animals at 14 days after TAC, as compared with WT animals. Finally, to investigate the hypertrophic signalling pathways activated in response to pressure overload, we examined both the CaMKII-HDAC4-MEF2 and calcineurin-NFAT signalling pathways 2 days after TAC in WT and Trpm4 cKO mice (Figure 6). This confirmed our previous finding that the CaMKII-HDAC4-MEF2 pathway was activated in response to TAC in WT mice (Yu et al., 2021), but also revealed the new finding of reduced activation of the CaMKII-HDAC4-MEF2 pathway after TAC in Trpm4 cKO animals.

Figure 6

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Previous studies reported that the TRPM4 current contributes to the mammalian atrial action potential (Simard et al., 2013) as well as to the notch and early repolarisation phases of the action potential in Purkinje cells (Hof et al., 2016), providing a potential link to cardiac arrhythmias (Guinamard et al., 2015; Hedon et al., 2021; Wang et al., 2018). Importantly, there is evidence that the TRPM4 channel is a critical modulator of ventricular remodelling in cardiac hypertrophy and heart failure (Frede et al., 2020; Jacobs et al., 2015; Kecskés et al., 2015; Mathar et al., 2014). Kecskés et al., 2015 reported that TRPM4 activation suppresses angiotensin II-induced cardiac hypertrophy, which is dependent on the activation of the calcineurin-NFAT pathway. It has been proposed that this is due to the Ca²⁺-dependent modulation of TRPM4 activity, which leads to membrane depolarisation in cardiomyocytes and thus reduces the driving force for Ca²⁺ influx via store-operated calcium entry (SOCE) through TRP canonical type 1 (TRPC1) and type 3 (TRPC3) ion channels (Kecskés et al., 2015; Wu et al., 2004).

To our knowledge, however, a role for TRPM4 in the LVH induced by mechanical pressure overload has not been demonstrated previously. We propose here that a mechanical stimulus, such as that exerted by TAC, is converted to downstream Ca²⁺ signalling via the activity of mechanosensitive ion channels in the plasma membrane. Although the mechanosensitivity of TRP-type ion channels is still the subject of debate (Cox et al., 2019; Gottlieb et al., 2008), mammalian TRP ion channels, including TRPM4, have recently been shown to be insensitive to membrane stretch (Constantine et al., 2016; Nikolaev et al., 2019). Therefore, TRPM4 does not appear to be the primary mechanosensor responding to pressure overload. It is more likely to be a secondary ionotropic receptor downstream of a Ca²⁺-permeable mechanosensitive ion channel, such as Piezo1 (Gnanasambandam et al., 2015; Syeda et al., 2016) or TRPV2/4 (Lieben and Carmeliet, 2012), that functions as the primary mechanoreceptor responding directly to pressure overload and thus initiating the hypertrophic response in TAC, which is not dependent on activation of the calcineurin-NFAT pathway (Yu et al., 2021).

Stimulated by the local Ca²⁺ influx through a Ca²⁺-permeable mechanosensitive ion channel, the Na⁺-permeable TRPM4 activity then could either induce reverse activity of the Na⁺/ Ca²⁺ exchanger through local Na⁺ loading (Conway and Koushik, 2001; Wang et al., 2018) or depolarise the cardiomyocyte cell membrane to stimulate voltage-gated Ca²⁺ channels. Such potential downstream ion channels include the L-type Ca²⁺ channels, which were reported to mediate hypertrophic cardiomyopathy (Viola and Hool, 2017), as well as the T-type Ca²⁺ channels whose splice variants were found to be regulated in rat LV hypertrophic hearts induced by aortic constriction (Cribbs, 2010). Either of these outcomes would lead to a high-amplitude increase in local Ca²⁺. Thus, as a Ca²⁺-dependent non-selective monovalent cation channel (Constantine et al., 2016; Launay et al., 2002; Nilius et al., 2003; Nilius et al., 2005), TRPM4 could contribute to TAC-induced LVH by modulating downstream voltage-gated Ca²⁺ ion channels or the Na⁺/Ca²⁺ exchanger (Figure 6).

In this study, we confirmed the involvement of TRPM4 in TAC-induced LVH using Trpm4 cKO mice. Despite identical TAC-induced increases in haemodynamic load in both WT and Trpm4 cKO mice, the latter displayed a significantly reduced LVH response. This is in contrast to the increased hypertrophy reported in angiotensin II-treated Trpm4 cKO mice that is mediated by the calcineurin-NFAT pathway (Kecskés et al., 2015). These differential effects of TRPM4 on angiotensin II-mediated (Kecskés et al., 2015) and TAC-induced LVH support our previous finding that these two hypertrophic stimuli are mediated by distinct signalling mechanisms. Thus, in agreement with the present findings, we showed previously that the CaMKII-HDAC4-MEF2 pathway, but not the calcineurin-NFAT signalling pathway, is activated in response to TAC-induced pressure overload (Yu et al., 2021). This is most likely because CaMKIIδ and calcineurin respond to different characteristics of intracellular Ca²⁺ signalling (De Koninck and Schulman, 1998; Dolmetsch et al., 1997). Whereas calcineurin activation requires a sustained increase in the resting intracellular Ca²⁺ concentration, CaMKIIδ activation is more sensitive to high-frequency/high amplitude Ca²⁺ oscillations (Colella et al., 2008; De Koninck and Schulman, 1998), which are known to occur with TAC-induced aortic constriction (Chen et al., 2011). CaM responds to this high-amplitude Ca²⁺ stimulus through the lower affinity Ca²⁺ binding site at its N-lobe (Evans and Shea, 2009; Saimi and Kung, 2002), which subsequently activates CaMKIIδ and thus stimulates the CaMKII-HDAC4-MEF2 pathway (Yu et al., 2021). Moreover, once activated, CaMKII has been shown to inhibit calcineurin activity (Kreusser et al., 2014; MacDonnell et al., 2009; Figure 6).

The high-frequency/high-amplitude Ca²⁺ oscillations characteristic of TAC and the consequent intermittent nature of the resulting TRPM4 activation, as distinct from the persistent activation associated with angiotensin II-induced hypertrophy, may mean that TAC-induced TRPM4 activation does not reduce the driving force for Ca²⁺ influx via SOCE that characterises the anti-hypertrophic action of TRPM4 in angiotensin II-induced hypertrophy (see above). Rather, in TAC-induced hypertrophy, TRPM4 acts as a second messenger, amplifying the small load-dependent Ca²⁺ signal produced by a mechanosensitive Ca²⁺-permeable ion channel. This would account for the pro-hypertrophic effect of TRPM4 in TAC-induced hypertrophy as distinct from its anti-hypertrophic effect in angiotensin II-induced hypertrophy.

In terms of the signalling pathway mediating pressure overload-induced LVH, we found that in Trpm4 cKO TAC hearts, the reduced LVH response was associated with significantly less activation of the CaMKII-HDAC4-MEF2 pathway, with reduced CaMKIIδ activation resulting in reduced nuclear export of HDAC4. Since nuclear HDAC4 inhibits MEF2A activity, a reduction in HDAC4 nuclear export would result in diminished MEF2A disinhibition and, given that MEF2A is a critical nuclear transcriptional regulator causing pathological cardiac remodelling, reduced hypertrophy development (Passier et al., 2000) as, indeed, observed here in Trpm4 cKO TAC hearts.

Decreased expression of TRPM4 channels in Trpm4 cKO animals likely modifies Ca²⁺-signalling, which directly regulates CaMKIIδ activation and its downstream pathway in response to TAC. Comparable with a study reporting that blockade of MEF2 acetylation can permit recovery from pathological cardiac hypertrophy without impairing physiologic adaptation (Wei et al., 2017), the lower concentration and reduced activity of MEF2A that we found in Trpm4 cKO TAC hearts suggest that inhibition of TRPM4 channels is potentially a viable therapeutic option for reducing pathological hypertrophy in response to pressure overload.

Interestingly, although the calcineurin-NFAT hypertrophic signalling pathway is not activated by TAC in WT hearts, it was partially activated in Trpm4 cKO TAC hearts, which manifested itself in the inhibition of GSK3β-mediated NFATc4 nuclear export and by an increase in GATA4. This may be explained by the reduction of the cytoplasmic CaMKIIδ in Trpm4 cKO TAC hearts, as CaMKII negatively regulates calcineurin activity (Kreusser et al., 2014; MacDonnell et al., 2009; Figure 6). It is notable, nevertheless, that the net effect of the loss of TRPM4 was a significant reduction in TAC-induced LVH, indicating that the direct effect of less activation of the CaMKII-HDAC4-MEF2 pathway in reducing hypertrophy development outweighed the indirect pro-hypertrophic effect resulting from blunting CaMKIIδ’s inhibition of calcineurin.

In summary, our study provides compelling evidence that TRPM4 plays an important role in pressure overload-induced pathological LVH, with diminished TRPM4 expression reducing TAC-induced hypertrophy. Furthermore, we demonstrated that TRPM4 is a likely component of a cardiac mechanotransduction process that activates the CaMKII-HDAC4-MEF2 pathway in response to TAC. It is likely that TRPM4 is activated by upstream primary mechanoreceptors, such as Piezo1 or TRPV2/4 channels, which provide the first step in this mechanotransduction pathway. These findings expand our understanding of the molecular mechanism underlying mechanical pressure overload-induced LVH. Moreover, our work provides new insights into possible treatment strategies for limiting pressure overload-induced pathological hypertrophy.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all the figures and tables.

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Author details

1. Yang Guo

   1. Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia
   2. Cardiac Physiology and Transplantation Division, Victor Chang Cardiac Research Institute, Sydney, Australia
   3. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - original draft

   Contributed equally with

   Ze-Yan Yu

   Competing interests

   No competing interests declared

2. Ze-Yan Yu

   1. Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia
   2. Cardiac Physiology and Transplantation Division, Victor Chang Cardiac Research Institute, Sydney, Australia
   3. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Writing - original draft

   Contributed equally with

   Yang Guo

   Competing interests

   No competing interests declared

3. Jianxin Wu

   Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia

   Contribution

   Data curation, Supervision, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Hutao Gong

   Cardiac Physiology and Transplantation Division, Victor Chang Cardiac Research Institute, Sydney, Australia

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

5. Scott Kesteven

   1. Cardiac Physiology and Transplantation Division, Victor Chang Cardiac Research Institute, Sydney, Australia
   2. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia

   Contribution

   Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Siiri E Iismaa

   1. Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia
   2. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia

   Contribution

   Data curation, Supervision, Methodology

   Competing interests

   No competing interests declared

7. Andrea Y Chan

   Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia

   Contribution

   Data curation, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

8. Sara Holman

   Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia

   Contribution

   Data curation, Validation, Methodology

   Competing interests

   No competing interests declared

9. Silvia Pinto

   1. Laboratory of Ion Channel Research, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium
   2. TRP Research Platform Leuven (TRPLe), Katholieke Universiteit Leuven, Leuven, Belgium

   Contribution

   Data curation, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

10. Andy Pironet

    1. Laboratory of Ion Channel Research, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium
    2. TRP Research Platform Leuven (TRPLe), Katholieke Universiteit Leuven, Leuven, Belgium

    Contribution

    Data curation, Investigation, Methodology

    Competing interests

    No competing interests declared

11. Charles D Cox

    1. Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia
    2. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia

    Contribution

    Conceptualization, Supervision, Investigation, Writing - original draft

    Competing interests

    No competing interests declared

12. Robert M Graham

    1. Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia
    2. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia

    Contribution

    Resources, Funding acquisition, Writing - original draft

    Competing interests

    No competing interests declared

13. Rudi Vennekens

    1. Laboratory of Ion Channel Research, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium
    2. TRP Research Platform Leuven (TRPLe), Katholieke Universiteit Leuven, Leuven, Belgium

    Contribution

    Resources, Funding acquisition, Methodology, Writing - original draft, Project administration

    Competing interests

    No competing interests declared

14. Michael P Feneley

    1. Cardiac Physiology and Transplantation Division, Victor Chang Cardiac Research Institute, Sydney, Australia
    2. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia
    3. Department of Cardiology, St Vincent’s Hospital, Sydney, Australia

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Validation, Methodology, Writing - original draft, Project administration

    For correspondence

    michael.feneley@svha.org.au

    Competing interests

    No competing interests declared

15. Boris Martinac

    1. Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia
    2. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Validation, Methodology, Writing - original draft, Project administration

    For correspondence

    b.martinac@victorchang.edu.au

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8422-7082

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