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TRPV4 expression in cardiomyocytes of the aged heart. (A) Example western blots of isolated cardiomyocyte homogenates of 3 Young (left 3 samples) and 3 Aged (right 3 samples) mice, probed with anti-TRPV4 (～100 kDa, upper) and anti-calsequestrin (CSQ, ～55 kDa, lower) antibodies. Densitometry profiles (background-subtracted using regions below bands of interest, with plots inverted for presentation, scale bar = 150 AU) are presented below images. (B) TRPV4:CSQ ratio (relative to levels in Young) in n = 8 Young mice (gray) and n = 6 Aged mice (blue). (C–E) Representative immunocytochemistry image of cardiomyocyte (C) or subcellular cardiomyocyte regions (D, E, microscope zoom) of Aged mice using antibodies for TRPV4 (C–E, red), α-tubulin (D, upper panel in green from perinuclear region) and Caveolin-3 (E, upper panel in green from sub-sarcolemmal region). Merge of green and red channels in D–E are presented in bottom panels, with yellow indicating colocalization. (F–G) Transmitted light (upper panels) and TRPV4 immunofluorescence (lower panels) images of fixed left ventricular tissue from an Aged Mouse (F) and a 68-year-old patient undergoing aortic valve replacement surgery (G). TRPV4 fluorescence intensity (black traces, from region denoted by bracket to left of images) is shown below images, alongside fluorescence intensity of IgG control obtained from duplicate section on each slide (gray traces). Intensity scale bar = 2000 arbitrary units. A similar staining pattern was observed in n = 13 additional left ventricular samples from both male and female patients, ages 57–87, with a range in ejection fraction between 34% and 76%. **P<0.01 Aged vs. Young.

Figure 1

TRPV4 is well-described as a Ca²⁺ influx channel responsive to hypoosmotic stress.16^,17 We, therefore, explored the functional role of TRPV4 on action-potential induced Ca²⁺ transient amplitude (fluo-4/AM) following hypoosmotic stress. Cardiomyocytes of Aged (Figure 2A and B), but not Young (see Supplementary material online, Figure S2), responded to hypoosmotic stress with an enhancement in Ca²⁺ transient amplitude. The hypoosmotic stress-induced increase in Ca²⁺ transient amplitude observed in Aged associated with faster Ca²⁺ reuptake kinetics (see Supplementary material online, Table S1), and these effects were prevented by the TRPV4 antagonist HC067047 (HC, 1?μmol/L). The TRPV4 antagonist did not affect Ca²⁺ transient parameters in Aged in the absence of hypoosmotic stress, and was without effect in Young under any experimental condition.

Figure 2

TRPV4 activation enhances Ca²⁺ transients via increased SR Ca²⁺ content and activation of RyR Ca²⁺ release. (A) Example Ca²⁺ transient traces (A) and summary data of Ca²⁺ transient amplitude (B) of electrically stimulated (0.5 Hz) cardiomyocytes of Aged mice. Ca²⁺ transients were examined under isomotic conditions (ISO: ～300 mOsm/L) and following transient hypoosmotic stress (HYPO: ～250 mOsm/L, followed by a return to isosmotic conditions for criterion measurement) in the absence (blue, open) and presence (black, closed) of the TRPV4 antagonist HC067047 (HC, 1?μmol/L). (C) Example line-scan Ca²⁺ spark images in a cardiomyocyte of Aged under isosmotic conditions (ISO, upper panel) and following hypoosmotic stress (HYPO, lower panel). Fluorescence profiles of Ca²⁺ sparks (from regions indicated by bars to left of images) are presented below images. (D) Summary data of Ca²⁺ spark frequency (sparks × 100 μm^?1 × s^?1) under control conditions (ISO) and following transient hypoosmotic stress (HYPO, followed by a return to isosmotic conditions for criterion measurement) in the absence (blue, open) and presence (black, closed) of the TRPV4 antagonist HC067047 (HC, 1 μmol/L). (E) Example fluo-5F action potential-induced Ca²⁺ transient (arrow) followed by 10 mmol/L caffeine-induced Ca²⁺ transient (bar) in cardiomyocytes of Aged under isosmotic conditions (ISO, left) and following hypoosmotic stress (HYPO, right). (F) Summary data of SR Ca²⁺ content (ΔF/F₀, _Caffeine) under control conditions (ISO) and following transient hypoosmotic stress (HYPO, followed by a return to isosmotic conditions for criterion measurement) in the absence (blue, open) and presence (black, closed) of the TRPV4 antagonist HC067047 (HC, 1 μmol/L). Two-way ANOVA revealed a significant interaction between osmotic conditions and antagonist treatment in Aged cardiomyocytes (P < 0.05). *P < 0.05 or **P < 0.01 HYPO vs ISO within control; ^#P < 0.05 or ^##P < 0.01 antagonist treatment vs. control within HYPO. Number of cells (n) from number of animals (N) are as follows: Aged ISO n = 23/N = 11; Aged ISO + HC: n = 18/N = 5; Aged HYPO: n = 36/N = 12; Aged HYPO + HC: n = 20/N = 6 (Ca²⁺ transients), Aged ISO n = 11/N = 9; Aged ISO + HC: n = 13/N = 4; Aged HYPO: n = 13/N = 8; Aged HYPO + HC: n = 13/N = 5 (Ca²⁺ sparks), and Aged ISO n = 12/N = 4; Aged ISO + HC: n = 10/N = 4; Aged HYPO: n = 15/N = 4; Aged HYPO + HC: n = 12/N = 4 (SR Ca²⁺ content).

Figure 2

In mouse ventricular cardiomyocytes the majority (～90%) of the Ca²⁺ transient is due to Ca²⁺ release from the SR via RyR release channels, which in turn is highly dependent on SR Ca²⁺ content.30 To assess the mechanisms by which TRPV4 augments Ca²⁺ transients in Aged following hypoosmotic stress, we monitored the frequency of spontaneous RyR Ca²⁺ sparks following rest from action-potential stimulation. Ca²⁺ spark frequency was low under isosmotic conditions (Figure 2C, upper) but increased following hypoosmotic stress (Figure 2C, lower). The increase in Ca²⁺ sparks observed following hypoosmotic stress was prevented by TRPV4 inhibition with HC067047 (Figure 2D). We next assessed if SR Ca²⁺ content contributes to enhanced SR Ca²⁺ release by monitoring the amplitude of the 10?mmol/L caffeine-induced Ca²⁺ transient in Aged cardiomyocytes loaded with the moderate-affinity Ca²⁺ indicator fluo-5F. The amplitude of the caffeine-induced fluo-5F Ca²⁺ transient (Figure 2E, denoted by bars) was elevated following hypoosmotic compared with isosmotic conditions, and this effect was prevented by TRPV4 inhibition with HC067047 (Figure 2F). Taken together, these data suggest that TRPV4 contributes to cellular and SR Ca²⁺ loading following hypoosmotic stress, with a subsequent increase in SR Ca²⁺ release during ECC.

Advancing age associates with numerous alterations in Ca²⁺ handling protein expression and function. We, therefore, evaluated the effect of increased TRPV4 expression on cardiomyocyte Ca²⁺ homeostasis, independent of advancing age, using a double-transgenic (DTg), Tg(αMHC-loxP-mCherrySTOP-loxP-TRPV4)1td × αMHC-MerCreMer mouse (Figure 3A) with tamoxifen-inducible, cardiac-specific TRPV4 expression. Induction of TRPV4 transgene expression in Young mice resulted in TRPV4 protein levels ～2-fold greater than those observed endogenously in Aged mice (Figure 3D). Consistent with observations in Aged C57BL/6 mice, cardiomyocytes of Young DTg mice exhibited an increase in Ca²⁺ transient amplitude following hypoosmotic stress, which was prevented by TRPV4 inhibition (Figure 3E and F). These effects were absent in Young DTg mice not fed tamoxifen, and absent in tamoxifen-fed αMHC-MerCreMer single transgenic mice (combined controls ΔF/F₀, ISO: 1.78?±?0.11 vs. ΔF/F₀, HYPO: 1.78?±?0.07).

Figure 3

Transgenic TRPV4 expression in Young mice enhances cardiomyocyte Ca²⁺ transients following hypoosmotic stress. (A) Schematic of double-transgenic (DTg) mouse line with the Tg(αMHC-loxP-mCherrySTOP-loxP-TRPV4)1td (upper) and αMHC-MerCreMer (lower) transgenes. In the absence of tamoxifen, the α-MHC promoter drives mCherry expression (with STOP sequence) within cardiomyocytes (B, upper, DTg:mCherry, overlay of mCherry fluorescence and transmitted light image). With tamoxifen treatment, Cre-recombinase excises the mCherry(STOP) sequence thereby eliminating mCherry expression (B, lower, DTg:Tam, overlay of mCherry fluorescence and transmitted light image), and inducing cardiac specific TRPV4 expression. (C) Example western blots of isolated cardiomyocyte homogenates of an Aged and DTg:Tam mouse, probed with anti-TRPV4 (～100 kDa, upper) and anti-calsequestrin (CSQ, ～55 kDa, lower) antibodies. (D) Summary data of TRPV4 expression in cardiomyocytes of DTg:Tam mice (green bar, n = 3) compared with levels observed endogenously in Aged mice (black bar, n = 6, data from Figure 1B). Expression level in Young C57BL/6 mice used for normalization is indicated by dashed line. ***P?<?0.001 DTg:Tam vs. Aged. (E–F) Example Ca²⁺ transient traces (E) and summary data of Ca²⁺ transient amplitude (F) of electrically stimulated (0.5 Hz) cardiomyocytes of Young DTg:Tam mice examined under control conditions (ISO: ～300 mOsm/L) and following transient hypoosmotic stress (HYPO: ～250 mOsm/L, followed by a return to isosmotic conditions for criterion measurement) in the absence (green, open) and presence (black, closed) of the TRPV4 antagonist HC067047 (1 μmol/L). Control groups associated with the MerCreMer system exhibited Ca²⁺ transient amplitudes (ΔF/F₀, HYPO: 1.78 ± 0.07 and ΔF/F₀, ISO: 1.78 ± 0.11) similar to those observed in Young C57BL/6 mice (see Supplementary material online, Figure S2). Two-way ANOVA revealed a significant interaction between osmotic conditions and antagonist treatment in Young DTg:Tam cardiomyocytes (P < 0.05). **P < 0.01 HYPO vs. ISO within control; ^##P < 0.01 antagonist treatment vs. control within HYPO. Number of cells (n) from number of animals (N) are as follows: Dtg:Tam 300 n = 13/N = 5; Dtg:Tam 300 + HC: n = 4/N = 3; Dtg:Tam 250: n = 17/N = 5; Dtg:Tam 250 + HC: n = 11/N = 3.

Figure 3

A pathological scenario associated with pronounced cardiomyocyte hypoosmotic stress is I/R injury.31 Therefore, we examined the role of TRPV4 in pressure development and contractility (dP/dt_Max) following global I/R in isolated perfused hearts of Young C57BL/6 vs. Young DTg mice, as well as Aged C57BL/6 vs. Aged TRPV4(?/?) mice. Hearts of Young C57BL/6 and Young DTg mice had similar baseline contractile performance (Figure 4, Control), and as expected each ceased pressure development during global ischaemia (Figure 4A and B, Ischaemia). However, in the early phases of reperfusion (<30?min), hearts of Young DTg mice exhibited enhanced contractile function vs. Young C57BL/6 mice (Figure 4, Reperfusion). Hearts of Aged C57BL/6 and Aged TRPV4(?/?) mice also had similar baseline contractile performance (Figure 5, Control) and ceased pressure development during global ischaemia (Figure 5A and B, Ischaemia). In early reperfusion (<30?min) hearts of Aged C57BL/6 mice exhibited enhanced contractile function vs. Aged TRPV4(?/?) mice (Figure 5, Reperfusion). In addition, the reperfusion-induced change in contractile function in Aged C57BL/6 was prevented by TRPV4 inhibition with 1?μmol/L HC067047 (see Supplementary material online, Figure S3). The enhanced contractile function observed in early reperfusion in Young DTg and Aged mice was not sustained 2?h after reperfusion (see Supplementary material online, Table S2). Taken together, these data identify cardiomyocyte TRPV4 as a novel mediator of enhanced contractile function early in I/R.

Figure 4

Transgenic TRPV4 expression enhances contractility following I/R in Young hearts. (A and B) Example traces of left ventricular pressure (upper) and rate of pressure change (dP/dt, lower) in hearts of Young (A) and Young tamoxifen-fed DTg (DTg:Tam) mice (B) under control conditions, during ischaemia, and following reperfusion. Contractility (dP/dt_Max, C) was elevated in hearts of Young tamoxifen-fed DTg mice (green, open) vs. Young mice (gray, closed) during reperfusion. Two-way repeated measures ANOVA revealed a significant interaction between conditions and genotype (P < 0.05). *P < 0.05 Young DTg:Tam vs. Young within reperfusion. Young n = 5; Young DTg:Tam n = 4.

Figure 4

Figure 5

TRPV4 enhances contractility following I/R in Aged hearts. (A and B) Example traces of left ventricular pressure (upper) and rate of pressure change (dP/dt, lower) in hearts of Aged (A) and Aged TRPV4(?/?) mice (B) under control conditions, during ischaemia, and following reperfusion. Contractility (dP/dt_Max, C) was elevated in hearts of Aged (blue, open) vs. Aged TRPV4(?/?) mice (black, closed) during reperfusion. Two-way repeated measures ANOVA revealed a significant interaction between conditions and genotype (P < 0.05). **P < 0.01 Aged TRPV4(?/?) vs. Aged within reperfusion. Aged C57BL/6 n = 5; Aged TRPV4(?/?) n = 5.

Figure 5

Excessive Ca²⁺ entry into cardiomyocytes is an established cause of cardiomyocyte necrosis and tissue damage following I/R.4 We, therefore, examined if TRPV4 inhibition reduces hypoosmotic stress-induced damage in isolated cardiomyocytes and I/R-induced cardiac damage in Langendorff-perfused hearts of Aged mice (corresponding to the population at high risk of myocardial infarction). In isolated cardiomyocytes of Aged mice, application of hypoosmotic stress resulted in 50% of cells exhibiting damage within one hour. When cardiomyocytes were treated with the TRPV4 antagonist HC067047 only 10% of cells exhibited significant damage (Figure 6A and B). Langendorff-perfused hearts were subjected to global I/R (45?min ischaemia, 2?h of reperfusion) and perfused with TTC to differentiate metabolically active vs. inactive tissue (Figure 6C), and under these conditions hearts of Aged mice exhibited a 20% loss of viable cardiac tissue. In contrast, hearts of Aged mice treated with the TRPV4 antagonist HC067047 exhibited only a 6% loss of viable tissue (Figure 6D). Taken together, these data are consistent with TRPV4 exerting an initial beneficial effect of enhanced Ca²⁺ cycling and contractility, with a secondary detrimental effect of excessive cardiomyocyte Ca²⁺ stress and damage following I/R in the aged heart.

Figure 6

Pharmacological TRPV4 inhibition prevents cardiomyocyte damage following hypoosmotic stress and cardiac damage following I/R in Aged mice. (A) Example images of cardiomyocytes of Aged prior to (ISO, t = 0) and 30 min following sustained hypoosmotic stress (HYPO, t = 30), in the absence (left) and presence (right) of the TRPV4 inhibitor HC067047 (HC, 1 μmol/L). (B) Percent of cardiomyocytes that exhibited irreversible contracture and damage within one hour following hypoosmotic stress in the absence (blue, open) and presence (black, closed) of HC. Values for both untreated and treated groups were obtained from each animal using a paired design (n = 5, animal indicated by solid line); *P<0.05 Aged + HC vs. Aged (paired t-test). (C) Representative images of TTC-stained (red colour = live tissue) cardiac sections of hearts of Aged mice following I/R in the absence (left) and presence (right) of HC (1 μmol/L). (B) Summary data of percent tissue death in sections of Aged mice in the absence (blue, open) and presence (black, closed) of HC. *P < 0.05 Aged + HC vs. Aged (unpaired t-test), n = 6 per group.

Figure 6

4. Discussion

4.1 TRPV4 channel expression in cardiomyocytes

The TRP ion channel superfamily has recently been appreciated as a prominent mediator of signal transduction within cardiomyocytes, and the present investigation adds to this emerging literature by providing the first description of the functional role of TRPV4 in cardiomyocytes of the aged heart. While organ-level TRPV4 expression in the heart is well-established, cell types known to express TRPV4 (including cardiac fibroblasts, vascular endothelial cells, and vascular smooth muscle cells) collectively far outnumber cardiomyocytes within the heart, and therefore, expression of TRPV4 in cardiomyocytes remains unclear.22^,32 Recent data in whole-heart homogenates of young mice subjected to pressure overload33 or ischaemia–reperfusion injury34^,35 show an increase in TRPV4 expression following pathological stimuli. While these findings suggest a change in TRPV4 expression in cardiomyocytes, changes in TRPV4 expression in other cell types (most notably fibroblasts36) may also underlie such findings. Our data reveal that with advancing age TRPV4 expression and function increases and exerts significant effects on cardiomyocyte Ca²⁺ homeostasis and contractile function. Further, our data in Young mice with transgenic TRPV4 expression indicate that increased TRPV4 expression in itself (i.e. independent of aging or disease processes) contributes to enhanced cardiomyocyte Ca²⁺ transients following hypoosmotic stress and hypercontractility following I/R.

4.2 TRPV4 enhances SR Ca²⁺ content and SR Ca²⁺ release during ECC

Although phylogenetically part of the TRP family of non-selective cation channels, TRPV4 functions primarily as a Ca²⁺ influx channel with a Ca²⁺:Na⁺ permeability ratio of ～6:1.15 Ca²⁺ influx via TRPV4 is substantial, due to both high single channel conductance (～90 pS) and Ca²⁺ signal amplification via co-operative gating behaviour.18 Increased TRPV4 activity may also lead to cytosolic Na⁺ accumulation, either directly through the channel or secondary to the elevation in Ca²⁺ via enhanced diastolic forward-mode NCX activity. During systole, elevated cytosolic Na⁺ favours reverse-mode NCX Ca²⁺ entry at the peak of the action-potential, which increases the amplitude of the Ca²⁺ transient,37 and such a mechanism has been proposed to underlie augmentation of ECC following TRPC channel activation.38 TRPV4 therefore, represents a significant mode of Ca²⁺ entry that, similar to other TRP channel members,9^,39 may shift net Ca²⁺ flux towards cell and SR Ca²⁺ accumulation (Figure 2F). In turn, increased SR Ca²⁺ enhances Ca²⁺ transient amplitude (Figure 2B) according to the fundamental relationship between SR Ca²⁺ content and SR Ca²⁺ release30 via SR luminal Ca²⁺ regulation of RyR activity.40 Although we only examined acute activation of TRPV4 in the present investigation, sustained TRP channel activity may also lead to phosphorylation of RyR by Ca²⁺/calmodulin-dependent protein kinase II,39 resulting in SR Ca²⁺ leak and a cellular phenotype of enhanced RyR Ca²⁺ spark frequency with no change (or even a decrease) in SR Ca²⁺ content. Therefore, TRP channel activation may lead to complex time-dependent changes, with initial enhancement of ECC followed by a secondary deterioration in function.

4.3 TRPV4-mediated contractile function and cardiomyocyte death following I/R

Excessive cardiomyocyte Ca²⁺ entry and adverse Ca²⁺ overload during I/R are classically believed to be pH-driven processes (for reviews, see refs41^,42). During ischaemia, cardiomyocytes shift energy production from oxidative phosphorylation to anaerobic glycolysis with concomitant lactic acid accumulation and intracellular acidification.43 During reperfusion the extracellular environment is rapidly restored creating a large outward H⁺ gradient which drives sequential Na⁺/H⁺ and Na⁺/Ca²⁺ exchange and an elevation in intracellular Ca²⁺ (Figure 7, right). Our data in Aged mice reveal a novel osmolarity-induced Ca²⁺ signalling pathway via TRPV4 in cardiomyocytes (Figure 7, left). We propose that during ischaemia, both the intracellular and extracellular environment gradually develop an increase in osmolarity31 yet osmotic stress on the cardiomyocyte membrane is minimal due to equilibration between the intracellular and extracellular compartments. During reperfusion, however, rapid washout of the extracellular fluid creates a marked hypoosmotic stress on the cardiomyocyte sarcolemma which activates TRPV4 and additional Ca²⁺ entry. The combination of pH-driven and osmolarity-driven Ca²⁺ entry leads to enhanced Ca²⁺ cycling and contractility in the early stages of reperfusion. However, the pronounced cardiomyocyte Ca²⁺ overload and excessive contractility render the aged heart highly susceptible to cardiomyocyte damage following I/R. Exacerbating the excessive plasma membrane Ca²⁺ entry is dysfunctional SR Ca²⁺ cycling due to oxidative modification of SERCA and RyR, which makes SERCA less effective at re-sequestering Ca²⁺ and leads to SR Ca²⁺ leak via RyR channels. Collectively, enhanced Ca²⁺ entry processes and dysfunctional SR Ca²⁺ handling create a vicious cycle of Ca²⁺ overload, during which mitochondria accumulate excessive amounts of Ca²⁺ within the mitochondrial matrix leading to mitochondrial permeability transition (MPT), rapid dissipation of the mitochondrial proton gradient, and mitochondrial depolarization.42 Further, Ca²⁺ overload-induced MPT ceases ATP production, leads to generation of reactive oxygen species, promotes mitochondrial swelling and rupture, and ultimately leads to cardiomyocyte necrosis and short-term cardiac dysfunction.4 Consistent with this working model, TRPV4-mediated Ca²⁺ entry was recently shown to mediate reactive oxygen species production, MPT, mitochondrial depolarization, and cell death in cultured H9C2 cells following hypoxia-reoxygenation challenge.20 Therefore, in the intact heart an initial increase in contractility is observed during early reperfusion due to a TRPV4-mediated enhancement of cardiomyocyte Ca²⁺ cycling (Figure 2) and contractile strength (Figure 5). However, the enhanced contractility is not maintained (see Supplementary material online, Table S2) due to cardiomyocyte death (Figure 6A and B) and cardiac damage (Figure 6C and D). In addition to the acute effects on cardiac dysfunction, cardiomyocyte death also activates an inflammatory response leading to a secondary phase of maladaptive remodelling and long-term cardiac dysfunction. This working model is consistent with the literature on short-term benefit/long-term harm with positive inotropes such as catecholamines. β-Adrenergic stimulation enhances Ca²⁺ cycling and contractility (a beneficial short-term effect) yet activation of this signalling pathway increases myocardial oxygen demand and leads to Ca²⁺-dependent cardiomyocyte necrosis.4 For this reason, β-inotropic support is only utilized to treat acute cardiogenic shock following myocardial infarction, and once patients are haemodynamically stable they are prescribed the reciprocal β-blocker treatment which provides long-term clinical benefit. TRPV4 inhibitors may therefore, represent a novel pharmacological approach to prevent excessive contractility, cardiomyocyte Ca²⁺ overload, and cardiomyocyte death following I/R.

Figure 7

Model of Cardiomyocyte Ca²⁺ overload following I/R in the aged heart. Ca²⁺ overload in the Aged heart induced by combined osmolarity-driven (left) and pH driven (right) processes. During ischaemia intracellular acidosis, blunted ATP generation, and oxidative stress alters the function of ion channels, transporters, and ATPases. Upon reperfusion accumulated metabolites in the extracellular fluid are eliminated, producing both hypoosmotic stress (left) and a large gradient for cellular H⁺ extrusion (right). Resulting TRPV4-mediated Ca²⁺ influx in combination with NHE/NCX coupled Ca²⁺-entry leads to Ca²⁺ overload, excessive contractility, and irreversible myocyte damage.

Figure 7

4.4 Acute vs. chronic effects of TRPV4 inhibition following I/R

Our data indicate that TRPV4 is expressed in cardiomyocytes of the aged heart and exerts effects on cardiomyocyte viability in the acute phase (min to h) of I/R. Interestingly, multiple recent investigations report that hearts of young mice exhibit a secondary increase in TRPV4 expression following I/R20^,35 that may contribute to cardiomyocyte apoptosis, myofibroblast differentiation, fibrosis,36^,44 adverse remodelling, and cardiac dysfunction.20^,35^,45 Taken together, these data suggest that pharmacological therapies which inhibit TRPV4 will exert beneficial effects in both the acute and chronic stages of I/R injury. However, our data highlight the need for immediate treatment with TRPV4 antagonists during reperfusion therapy to prevent acute Ca²⁺-dependent cardiomyocyte damage, and the therapeutic window to obtain maximum clinical benefit may be shorter than previously reported.

4.5 Study limitations

Cardiac I/R associates with complex changes in tissue osmolarity, with a gradual increase in osmotically active particles in both the intracellular and extracellular compartments, followed by reperfusion and a rapid reversion of the extracellular environment to normal osmotic conditions with associated hypoosmotic stress on the cardiomyocyte. Given the difficulty in precisely replicating this form of stress in the isolated cell environment, we utilized a defined reduction in osmotically active particles in the extracellular solution to induce hypoosmotic stress on cardiomyocytes. A global I/R protocol was used to examine contractile function and tissue damage in isolated perfused hearts. Unfortunately, this protocol associated with frequent supraventricular tachycardia and other complex arrhythmias, which precluded rigorous classification and quantification of ventricular arrhythmia burden in our experiments. Thus, while Ca²⁺-dependent arrhythmia is well-established following I/R,41 the specific role of TRPV4 in ventricular arrhythmia post I/R remains to be appropriately tested. Heteromeric assembly of TRPV4 with other TRP channel family members is well-established,46 including with members known to be expressed in cardiomyocytes such as TRPV2 and TRPC6. It is, therefore, plausible that TRPV4 inhibition may prevent ion flux through multiple TRP channels, as has been shown with dominant-negative TRPC expression in cardiomyocytes.10^,39 TRPV4 antagonists exhibit excellent pharmacological properties in vivo47 and are currently in clinical trials to improve pulmonary function in heart failure (NCT02119260, NCT02497937). Heteromeric assembly of TRPV4 with other TRP channel members would therefore, be advantageous, as it would allow TRPV4 inhibitors to prevent adverse effects of several TRP channel family members.

5. Conclusions

TRPV4 is now appreciated as a critical cellular signal integrator. Our working model is that increased TRPV4 expression in the aged heart is a physiological adaptation that enhances Ca²⁺ cycling and contributes to processes such as cardiac mechanotransduction, cell volume regulation, or Ca²⁺-dependent hypertrophic remodelling. However, during I/R, physiological TRPV4-mediated Ca²⁺ signal transduction transitions into pathological Ca²⁺ signal overload. When combined with pH-driven Ca²⁺ entry processes, TRPV4-mediated Ca²⁺ entry leads to excessive contractility and adverse tissue damage. Over 50% of all hospital admissions and 80% of deaths due to myocardial infarction occur in elderly individuals. TRPV4 may therefore, represent a novel ion channel target to prevent tissue damage and mortality following myocardial infarction.

Acknowledgements

The authors would like to thank the laboratory of Dr Michael Davis for providing PDGFRαGFP mice. The Trpv4-mCherry fusion mice were generated by the University of Missouri Animal Modeling Core. The authors would like to thank G. Ellison for contributions in equipment design and construction.

Conflict of interest: none declared.

Funding

This work was supported by research funding from NIH R01HL094404 (C.P.B.), R01HL116525 (M.K.), K01AG041208 (T.L.D.), and R01HL136292 (T.L.D.). Additional project support was obtained from a University of Missouri System Research Board grant (T.L.D.) and a University of Missouri School of Medicine Research Fellowship (D.P.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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