TAC induces identical levels of LVH in WT and Piezo1P1-tdT/P1-tdT mice
We hypothesized that Piezo1 is involved in the signalling cascade that drives pathological LVH in response to pressure overload. To test this hypothesis, we employed Piezo1P1-tdT/P1-tdT mice that expressed a Piezo1-tdTomato fusion protein from the Piezo1 locus12. This reporter mouse allowed us to use Piezo1 fusion proteins to probe Piezo1 expression using a specific mCherry antibody. We took this approach because there are no specific mouse anti-Piezo1 antibodies available commercially. We performed transverse aortic constriction (TAC) on Piezo1P1-tdT/P1-tdT mice and their wild type littermates (WTLs).
TAC increased LV systolic pressure (LVSP) by ~57 mmHg in both WTLs and Piezo1P1-tdT/P1-tdT mice, when compared with their respective sham-operated controls at 14 days (Table 1, both p < 0.001). This significant pressure overload with TAC resulted in enlarged hearts (Table 1, Supplementary Fig. 1A) and significant LVH after 14 days in both groups, when compared with their sham-operated controls. The degree of LVH did not differ between WTLs and Piezo1P1-tdT/P1-tdT mice, whether LVH was assessed by echocardiographically-determined LV mass, wall thickness (h), or wall thickness to chamber radius ratio (h/r), or by postmortem LV weight (LVW), whether normalized to body weight (LVW/BW) or tibial length (LVW/TL) (Table 1). Consistent with the development of pathological LVH, TAC was associated with increased cardiac fibrosis (p < 0.001, Table 1, Supplementary Fig. 1B) and enhanced collagen III (Col3a1) expression (p < 0.001, Table 1) in both WTLs and Piezo1P1-tdT/P1-tdT mice.
Notably, TAC-induced LVH at 14 days was not associated with any evidence of LV dysfunction in either group: there were no significant changes in heart rate (HR), echocardiographically-determined LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), LV ejection fraction (LVEF), cardiac output (CO), or dP/dtmax, dP/dtmin, lung weight or lung weight to body weight ratio (Table 1), indicating that our TAC model is a model of pressure overload LVH without ventricular decompensation or heart failure, as reported previously6, 7.
Early markers of LVH induction in WT and Piezo1P1-tdT/P1-tdT mice
Consistent with the early induction of hypertrophic signalling after TAC6, 7, gene expression of atrial natriuretic peptide (ANP, Nppa), brain natriuretic peptide (BNP, Nppb) and α-skeletal actin (α-SA, Acta1) was increased significantly 48 hours after TAC, preceding the development of significant LVH (supplementary Table 1), in both whole LV tissue and isolated LV cardiomyocytes, with no significant differences between WTLs and Piezo1P1-tdT/P1-tdT hearts (Table 2). The increased expression of these genes in whole LV tissue and isolated LV cardiomyocytes persisted 14 days after TAC in WTLs and Piezo1P1-tdT/P1-tdT mice (Table 2).
Pressure overload induces Piezo1 upregulation
Under baseline conditions, Piezo1 mRNA levels were low in isolated LV cardiomyocytes when compared to whole heart tissue, brain, aorta and lung (Fig. 1A). This is consistent with previous reports9, 11. We used the Piezo1-tdTomato fusion protein to measure Piezo1 protein levels. Western blots demonstrated a clear band at ~320 kDa, indicative of the Piezo1-tdTomato fusion protein, in both whole heart tissue and isolated cardiomyocytes (Fig. 1B). Under baseline conditions, Piezo1 protein levels were low in isolated cardiomyocytes, particularly when compared with the aorta and lung (Fig. 1C).
Two days after TAC, however, Piezo1 mRNA expression increased significantly, ~2-fold in LV tissue (p < 0.01) and ~6-fold in isolated LV cardiomyocytes (p < 0.001, Fig. 1D), when compared with sham-operated hearts. The upregulation of Piezo1 mRNA expression was not maintained 14 days after TAC, by which time mRNA expression was similar to that in sham-operated controls in both LV tissue and isolated cardiomyocytes (Fig. 1D). In contrast, Piezo1 protein levels did not increase significantly in either LV tissue or isolated cardiomyocytes 2 days after TAC (Fig. 1E and F), but did increase significantly 14 days after TAC in both LV tissue (~1.6-fold, p < 0.001) and isolated cardiomyocytes (~1.4-fold, p < 0.01) (Fig. 1E and F).
Targeted deletion of Piezo1 from cardiomyocytes in the adult heart
To further investigate whether Piezo1 plays an important role in the induction of LVH secondary to pressure overload, we generated an inducible cardiomyocyte-specific Piezo1 knockout mouse (Piezo1 KO) that permitted targeted deletion of Piezo1 from cardiomyocytes in adult mice (8 weeks old) (Fig. 2A, B). Tamoxifen inducible α-MHC-MerCreMer transgenic mice (Cre transgene under control of the α-myosin heavy-chain (Myh6) promoter15) were crossed with Piezo1fl/fl mice, and offspring backcrossed until P1fl/flMCM+/- were generated (see Methods and Fig. 2B, C). To account for the potential of nonspecific Cre-recombinase mediated cardiotoxicity16, P1wt/wtMCM+/- mice were designated as controls for Cre. P1fl/flMCM+/- male mice aged 8-10 weeks were indistinguishable in cardiac function and anatomical parameters from age- and sex-matched P1wt/wtMCM+/- and P1wt/wtMCM-/- control mice (Supplementary Fig. 2).
To establish a dosing regimen of tamoxifen that maximized Cre-recombinase activity at the Piezo1 locus but minimized tamoxifen-induced cardiotoxicity16, 17, we injected different concentrations of tamoxifen into 8-week-old male P1fl/flMCM+/- and P1wt/wtMCM+/- mice on 3 consecutive days, and allowed 10 days for them to recover. We injected the same volume of peanut oil into P1fl/flMCM+/- mice as the treatment control. We observed that tamoxifen induced Cre recombinase activity in a dose-dependent manner (Supplementary Fig. 3A - C). Piezo 1 deletion was observed in the cardiomyocytes of mice injected with tamoxifen but not peanut oil. P1fl/flMCM+/- mice treated with tamoxifen at 30 mg/kg/d exhibited normal heart structure and function when compared with mice treated with peanut oil (Supplementary Table 2). Higher doses of tamoxifen, 50 mg/kg/d or 100 mg/kg/d, caused LV dilatation with impaired contraction (Supplementary Table 2). Consequently, a tamoxifen dose of 30 mg/kg/d was used in subsequent experiments.
As treatment for three consecutive days did not result in a satisfactory level of Piezo1 excision we increased the number of consecutive days of tamoxifen dosing. Ultimately, tamoxifen at 30 mg/kg/d for six consecutive days produced efficient inducible deletion of Piezo1 in P1fl/flMCM+/- mice when measured ten days after the last injection with no impact on cardiac function (Fig 2). We confirmed successful Cre-recombination using PCR amplification of cardiac genomic DNA18. As shown in Figure 2C, Cre-mediated recombination of Piezo1 was detected only in the heart, with no excision identified in liver or lung tissues. A stronger signal was identified in isolated cardiomyocytes when compared to whole heart tissue from tamoxifen-treated P1fl/flMCM+/- mice, and no excision was evident in cardiomyocytes isolated from control mice treated with peanut oil (Fig.2 C).
Congruent with these results, and consistent with previous findings of the relative efficiency for the αMHC-MerCreMer construct15, 19, expression of Piezo1 mRNA was reduced by approximately 78% in isolated cardiomyocytes from tamoxifen treated P1fl/flMCM+/- mice (Piezo1 KO mice, p < 0.001) when compared with mice injected with peanut oil (Fig. 2C, D). These findings confirm that tamoxifen 30 mg/kg/d for 6 days induced specific deletion of Piezo1 from cardiomyocytes.
Next, we assessed whether tamoxifen-induced deletion of Piezo1 in cardiomyocytes had any impact on baseline cardiac function and structure. To do this, we treated P1fl/flMCM+/- mice with the same tamoxifen dose (30 mg/kg/d) as a control for Cre expression (termed α-MHC-MCM+/- mice). We performed echocardiographic measurements 10 days after mice received their last injection of tamoxifen for Piezo1 KO mice, α-MHC-MCM+/- mice and P1fl/flMCM+/- control mice (peanut oil injected). The results from this analysis showed that there were no significant differences in body weight (Fig. 2E), heart rate (Fig. 2F), cardiac function (Fig2. G-J) or left ventricular morphology between any of the three groups (Fig. 2 K, L).
Cardiomyocyte-specific deletion of Piezo1 inhibits the hypertrophic response to pressure overload
TAC or sham surgery was performed on Piezo1 KO mice, α-MHC-MCM+/- mice and P1fl/flMCM+/- control mice. After 14 days, body weight, heart rate, LV end-diastolic and end-systolic volumes, ejection fraction and dP/dt were not significantly different between TAC or sham-operated animals, or between the three genotypes (Fig.3, Supplementary Table 3). Importantly, TAC induced the same increase in LV systolic pressure in all three genotypes (Fig. 3C). Echocardiographic indices of LVH - LV wall thickness to chamber radius ratio (h/r) and LV mass – increased significantly (both p < 0.001) after TAC in both α-MHC-MCM+/- mice and P1fl/flMCM+/- control mice (Fig. 3E, F), but these changes were absent in Piezo1 KO mice after TAC (Fig. 3E, F). Similarly, post mortem indices of LVH – heart weight and LV weight, whether normalised to body weight or tibial length – increased very significantly (all p < 0.001) 14 days after TAC in both α-MHC-MCM+/- mice and P1fl/flMCM+/- control mice, but Piezo1 KO mice exhibited significant inhibition of the hypertrophic response to TAC (Fig. 3G-K): for example, the average increase in the LV weight/tibial length ratio after TAC was 62% lower in Piezo1 KO mice (p < 0.001).
Cardiomyocyte-specific deletion of Piezo1 inhibits myocardial fibrosis in response to pressure overload
Pathological LVH is associated with cardiac fibrosis, with upregulated collagen expression and deposition. We evaluated interstitial cardiac fibrosis in response to pressure overload 14 days after TAC in Piezo1 KO hearts and α-MHC-MCM+/- hearts using Masson’s trichrome staining (Fig. 3L). There was increased interstitial cardiac fibrosis in α-MHC-MCM+/--TAC hearts when compared with their sham controls (p < 0.001), but there was no increase in cardiac fibrosis in Piezo1 KO hearts after TAC (Fig. 3L, M). Consistent with these findings, Collagen III (Col3a1) mRNA expression increased ~6-fold in α-MHC-MCM+/--TAC hearts when compared with their sham controls (p < 0.001), but a more attenuated response was observed in Piezo1 KO hearts after TAC when compared with sham-operated hearts (~2-fold; p < 0.05, Fig. 3N) and with αMHC-MCM+/- TAC-operated hearts (p < 0.001, Fig. 3N).
Cardiomyocyte-specific deletion of Piezo 1 inhibits early markers of induction of hypertrophy in response to pressure overload
Although LVH had not yet developed 2 days after TAC (Supplementary Fig. 4), induction of hypertrophic signalling in α-MHC-MCM+/- hearts was already evident at this time, as was apparent from 5 to10-fold increases in gene expression of ANP (Nppa), BNP (Nppb) and α-SA (Acta1) (all p < 0.001). However, this early response of markers of hypertrophic signalling was completely absent in Piezo1 KO hearts 2 days after TAC (Fig. 4A). The increased expression of these hypertrophy-associated genes was maintained 14 days after TAC in α-MHC-MCM+/- hearts (~4 to 10-fold, all p < 0.001), while an attenuated late response was observed in Piezo 1 KO mice 14 days after TAC (~2 to 5-fold, p < 0.001 for both Nppa and Nppb, not significant for Acta1, Fig. 4B).
Cardiomyocyte-specific deletion of Piezo1 prevents activation of the CaMKII-HDAC4-MEF2 hypertrophic signalling pathway in response to pressure overload
The cytoplasmic and the nuclear fractions of LV tissue were separated as described in the Materials and Methods. Purity of the isolated fractions was confirmed by western blot analysis using antibodies against marker proteins specific for cytoplasmic (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) and nuclear (Histone H2B) fractions (Supplementary Fig. 5).
As expected, α-MHC-MCM+/- mice exhibited strong activation of the CaMKII-HDAC4-MEF2 hypertrophic signalling pathway 2 days after TAC (Fig. 5), consistent with our previous findings in TAC-induced hypertrophy6, 7. The hallmark of this activation is increased levels of both total and activated CaMKII, which is auto-phosphorylated at threonine 287 (p-CaMKII)20, 21, and the resultant increase in the cytoplasmic to nuclear ratio of HDAC4 (p < 0.01), indicating nuclear export of HDAC4, with consequent de-repression of MEF2A (p < 0.001, Fig. 5).
Remarkably, Piezo1 KO mice failed to exhibit any evidence of activation of the CaMKII-HDAC4-MEF2 hypertrophic signalling pathway 2 days after TAC: the findings in Piezo1 KO mice 2 days after TAC were indistinguishable from those in their sham-operated controls (Fig. 5). These data indicate that Piezo1 is essential for activation of the CaMKII-HDAC4-MEF2 hypertrophic signalling pathway in response to pressure overload-induced by TAC.
Cardiomyocyte-specific deletion of Piezo1 is associated with activation of the calcineurin-NFAT hypertrophic signaling pathway in response to pressure overload
As expected, α-MHC-MCM+/- mice exhibited no evidence of activation of the calcineurin-NFAT hypertrophic signalling pathway 2 days after TAC (Fig. 6), consistent with our previous findings in TAC-induced hypertrophy6, 7. One explanation for this finding is that pressure overload activates CaMKII and activated CaMKII inhibits calcineurin activation22. Because Piezo1 KO mice exhibited no evidence of CaMKII activation in response to pressure overload, yet they exhibited incomplete inhibition of LVH, we postulated that the residual LVH was driven by calcineurin activation in the absence of its inhibition by CaMKII. The results obtained in Piezo1 KO mice 2 days after TAC supported this hypothesis: the hallmark of calcineurin activation, an increase in the nuclear to cytoplasmic NFAT ratio, indicating translocation of NFAT to the nucleus due to dephosphorylation by activated calcineurin, was clearly evident in Piezo1 KO mice 2 days after TAC (p < 0.01, Fig. 6A, B) but absent in both sham-operated controls and α-MHC-MCM+/- mice subjected to TAC.
Piezo1 is upstream of changes in Ca2+ handling proteins in response to pressure overload
We have demonstrated recently that the Ca2+-activated ion channel TRPM4 plays an important role in the activation of the Ca2+-calmodulin dependent kinase, CaMKII, and thus the CaMKII-HDAC4-MEF2 hypertrophic signalling pathway7, but TRPM4 is neither stretch-activated nor Ca2+-permeable, whereas Piezo1 is both stretch-activated and Ca2+-permeable. To investigate whether Piezo1 can modify the expression of proteins important in cardiomyocyte Ca2+ handling, we probed the expression of TRPM4, the sodium-calcium exchanger (NCX1) and the T-type Ca2+ channel (Cav3.2) in response to LV pressure overload.
α-MHC-MCM+/- hearts showed significantly reduced expression of Trpm4 mRNA and TRPM4 protein 2 days after TAC (Fig. 7A, E and F, both p < 0.01), replicating our previous findings in wild type mice7, but Piezo1 KO mice exhibited no change in TRPM4 mRNA 2 days after TAC (Fig. 7A, E and F), indicating that Piezo1 is upstream of TRPM4, and mediates the response of TRPM4 to pressure overload.
Conversely, the upregulation of Piezo1 mRNA expression 2 days after TAC in wild type hearts (p < 0.001, Fig. 7B) was not abolished in TRPM4 KO hearts (p < 0.01) 2 days after TAC, confirming that TRPM4 is downstream of Piezo1 in the response to pressure overload. Nevertheless, the magnitude of upregulation of Piezo1 mRNA 2 days after TAC was diminished significantly in TRPM4 KO mice (p < 0.001, Fig. 7B), suggesting that TRPM4 plays a significant role in the feedback regulation of Piezo1 in response to pressure overload.
α-MHC-MCM+/- hearts exhibited significant increases in both NCX1 mRNA (Slc8a1) (Fig. 7C) and protein expression 2 days after TAC (Fig. 7E and G, both p < 0.01). In contrast, Piezo1 KO hearts exhibited no change in NCX1 mRNA or protein levels 2 days after TAC (Fig. 7C, E and G), indicating that Piezo1 is upstream of NCX1, and mediates the response of NCX1 to pressure overload.
α-MHC-MCM+/- hearts exhibited significant decreases in the gene expression of the T-type calcium channel (Cacna1h) 2 days after TAC (p<0.01), which was abolished in Piezo1 KO hearts (Fig. 7D), but the absence of any change at the protein level (Cav3.2) 2 days after TAC in α-MHC-MCM+/- hearts (Fig. 7H) casts some doubt on the functional significance of the T-type calcium channel in mediating the response to pressure overload governed by Piezo1.