Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction

Abstract

Rationale

The cellular and molecular basis for post myocardial infarction (MI) structural and functional remodeling is not well understood.

Objective

To determine if Ca²⁺ influx through transient ^receptor potential (canonical) (TRPC) channels contributes to post-MI structural and functional remodeling.

Methods and Results

TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca²⁺ entry. Cardiac myocyte specific expression of a dominant negative (dn: loss of function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes (AFMs) to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 over expression in AFMs induced calcineurin (Cn)-Nuclear Factor of Activated T cells (NFAT) mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in AFMs increased rested state contractions and increased spontaneous sarcoplasmic reticulum (SR) Ca²⁺ sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady state pacing, likely due to enhanced SR Ca²⁺ leak.

Conclusions

Ca²⁺ influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function.

INTRODUCTION

Cardiac systolic stress is increased in cardiovascular diseases such as hypertension and myocardial infarction (MI) and this requires an increase in contractile Ca²⁺. Persistent pathological stress usually results in a Ca²⁺ dependent pathological hypertrophy with Ca²⁺ related contractility defects. Abnormal contractile Ca²⁺ with depressed contractility reserve are hallmarks of cardiac hypertrophy and heart failure¹, but this contractile Ca²⁺ does not appear to be the source for activation of the signaling pathways that causes pathological hypertrophy. Recent data suggest that separate pools of myocyte “signaling” and “contractile” Ca²⁺ are involved in the induction of hypertrophy². The source and cellular location of the signaling Ca²⁺ is still not clearly defined. The present study explores the hypothesis that the expression of transient receptor potential (TRP) channels are induced after MI and Ca²⁺ influx through these channels within specific microdomains is necessary for the development of pathological hypertrophy, as well as for affecting contractility reserve that ultimately contributes to impaired pump function of the diseased heart^{3, 4}.

The function of the TRP family of channels is not well understood in the heart but it has been implicated in contributing to the initiation of pathological cardiac remodeling^5-8. TRP channels are a class of nonselective cation influx channels that are grouped into 7 families^{6, 9} and are present in many different cell types¹⁰. The TRP (Canonical) family includes 7 isoforms (TRPC1 to 7) that have been divided into 2 general subfamilies based on structural and functional similarities: TRPC1/4/5 and TRPC3/6/7⁶. In general, TRPC3/6/7 are activated by diacylglycerol generated by G protein-coupled receptors (GPCRs)/Gαq/phospholipase C signaling¹¹ while TRPC1/4/5 can be activated by stretch or depletion of intracellular Ca²⁺ stores (store-operated Ca²⁺ entry, SOCE)^{12, 13}, however TRPC6 has also been implicated as a mechano-sensing isoform as well¹⁴. Functional TRPC channels are formed as tetramers of individual 6-transmembrane spanning subunits. Interestingly, the channels can be homomeric or heteromeric assemblies with oligomerization occurring within and between subfamilies or beyond the TRPC family altogether (i.e. TRPCs can oligomerize with TRPVs and TRPMs)^15-18.

TRPC channels are expressed at very low levels in normal adult cardiac myocytes, but expression and activity of select isoforms appear to be increased in pathological hypertrophy and heart failure^{5, 7, 8, 19}. TRPC channels have been suggested as initiators of Ca²⁺-dependent signaling that leads to pathological cardiac remodeling, hypertrophy and failure^{5, 6}. Transgenic (TG) cardiac specific overexpression of TRPC3 or TRPC6 channels in mice causes re-expression of fetal genes, myocyte hypertrophy and activation of apoptotic signaling^{7, 20, 21}. The pro-hypertrophic effects of TRPC channels have also been shown in vitro in cultured cardiomyocytes^{5, 22}. Studies involving loss of TRPC function suggest a necessary role for these channels in pathological hypertrophy. TRPC3 inhibition with the inhibitor Pyr3 blocks cardiac hypertrophy in mice subjected to pressure overload²³ and this finding has been supported by data in gene-deleted mice (TRPC1⁸ and TRPC3/6²⁴) and in mice expressing dominant-negative mutants of select channels (dnTRPC3,4,6)²¹. Interestingly, mice expressing dnTRPC4 also inhibited the activity of the TRPC3/6/7 subfamily in the heart which suggests that TRPC 1/4/5 and 3/6/7 subfamilies function in coordinated complexes, at least when overexpressed²¹. The present study takes advantage of the dnTRPC strategy to define the role of TRPCs in post-MI structural and functional remodeling.

Ca²⁺ influx through TRPC channels was shown to activate the Ca²⁺ sensitive phosphatase calcineurin (Cn) that initiates diverse intracellular responses through its downstream transcriptional effector, nuclear factor of activated T cells (NFAT) ^{12, 13, 15, 22}. Cn-NFAT signaling in the heart is a well-known prohypertrophic pathway that is both necessary and sufficient for pathological growth³. Activation of this signaling cascade is thought to be the primary mechanism through which TRPC channels regulate cardiac hypertrophy. A recent in vitro study from our group suggests that TRPCs and L-type Ca²⁺ channels (LTCC) work in a coordinated fashion to activate Cn-NFAT signaling²². Additional studies from our group showed that a subpopulation of LTCCs localized specifically to caveolae membrane signaling microdomains are involved in pathological hypertrophic signaling²⁵. The present study explores the hypothesis that TRPCs and LTCCs function as essential partners in these caveolae signaling microdomains where their activity initiates hypertrophic Cn-NFAT signaling after MI.

The role of TRPC channels within excitation-contraction (EC) coupling microdomains has not been clearly defined. There are data in mice associating increased TRPC activity with reduced contractility^{7, 20} and loss of TRPC function with increased contractility²¹, but the mechanisms underlying these effects are not understood. The present study explores the hypothesis that Ca²⁺ influx through TRPC channels within EC coupling microdomains results in reduced contractility reserve after MI.

METHODS

Please refer to the extended Materials and Methods section in the Online Data Supplement for detailed methods. In brief, adult mouse and feline myocytes were isolated as extensively described^26-29. TRPC-mediated Ca²⁺ entry²⁰ and Ca²⁺ spark activity were measured in unpaced myocytes loaded with Fluo-4 while pacing protocols were implemented for fractional shortening and Ca²⁺ transient contractility studies^26-29. NFAT translocation studies were performed in AFMs using adenoviral mediated expression of NFAT-GFP^{22, 25} and immunoprecipitations, sucrose density gradients, and Western blot analysis were used for membrane localization²⁵ and phosphorylation³⁰ studies. Animal procedures were approved by the Temple University Institutional Animal Care and Use Committee. We induced MI in mice by permanent occlusion of the left main coronary artery (LCA) as previously described³¹ and animals were monitored over the course of the study using in vivo echocardiography (ECHO)³⁰.

RESULTS

TRPC channel expression and activity is induced after MI

Individual TRPC channels are expressed at low levels in normal adult heart, but expression and activity of select isoforms are upregulated in pathological stress conditions^{3, 6-8, 19}. We induced MI in mice as previously described³¹ and measured the abundance of individual TRPC channel mRNA by RT-PCR. MI resulted in the significant induction of hypertrophic gene markers and TRPC1/3/4/6 isoforms in mice 1-, 2- and 6-weeks post-MI compared to sham animals (Figure 1A).

Figure 1. Myocardial infarction induces TRPC channel expression and activity in mice and over-expression of TRPC channels in feline myocytes leads to increased membrane Ca²⁺ influx.

A, RT-PCR shows an up-regulation of TRPC1/3/4/6 channel isoforms at 1-, 2-, and 6-weeks post-MI along with the activation of the fetal gene program. B-D, TRPC-mediated Ca²⁺ entry in isolated myocytes from sham mice (left) or 1-week (B), 2-week (C), or 6-week (D) post-MI mice (right) in the presence of the TRPC channel agonist OAG (10umol/L) and the SERCA inhibitor CPA (5umol/L). Where indicated, the TRPC antagonists SKF-96365 (5umol/L), GSK503A (GSK, 10umol/L), Pyr10 (3umol/L) or the LTCC inhibitor nifedipine (Nif, 10umol/L) were used. E, AFMs infected with the indicated adenoviruses and assayed for TRPC-mediated Ca²⁺ entry. p<0.05 was considered significant (ns, p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001 vs. sham).

TRPC-mediated Ca²⁺ influx was determined using a TRPC-mediated Ca²⁺ entry bioassay. In most cell types, the depletion of intracellular Ca²⁺ stores leads to the activation of store-operated Ca²⁺ entry (SOCE) through defined channel complexes that include STIM and Orai and potentially TRPC channels on the plasma membrane³². Although the role of TRPC channels in SOCE in cardiac myocytes is not well defined, SR Ca²⁺ depletion of isolated myocytes followed by the re-introduction of Ca²⁺ in the presence of TRPC channel activator 1-oleoyl-2-acetyl-sn-glycerol (OAG), a stable cell permeable analog to the known TRPC agonist diacylglycerol, is an approach that has been used to assess TRPC-mediated Ca²⁺ entry^{6, 20}. Myocytes isolated from mice 1-, 2-, or 6-weeks post-MI showed substantial TRPC-mediated Ca²⁺ entry while myocytes from sham animals showed no detectable activity (Figure 1B-D). Similar results were seen in these MI myocytes in response to angiotensin II (Online Figure IA). The specificity of TRPC-mediated Ca²⁺ entry in MI myocytes was validated by inhibition with the pan TRPC antagonist SKF-96365 or the TRPC3/6 specific inhibitor GSK503A²⁴ (Figure 1B-D). TRPC-mediated Ca²⁺ entry was not inhibited by the LTCC antagonist nifedipine, documenting that this Ca²⁺ entry is independent of LTCC-mediated Ca²⁺ entry. Controls were also performed in cells incubated with CPA alone which resulted in a transient Ca²⁺ entry in 6-week MI myocytes but no detectable entry in sham myocytes (Online Figure IB) and also with myocytes treated with OAG alone (Online Figure IC), which resulted in Ca²⁺ entry in MI myocytes only and lead to spontaneous contractions.

Adult feline ventricular myocytes expressing TRPC channels have increased membrane Ca²⁺ influx

To further characterize the properties of TRPC channels in adult cardiac myocytes we used cultured isolated adult feline myocytes (AFMs) because their electrophysiological and Ca²⁺ regulatory properties more closely resemble those of human myocytes (in comparison to rodent myocytes)³³. AFMs survive in culture without the use of drugs that reduce Ca²⁺ influence and overload. This allows for manipulation of protein expression using adenoviral vectors in an adult myocyte that maintains stable electrical and mechanical properties^{27, 34-36}.

We performed TRPC-mediated Ca²⁺ entry measurements in AFMs infected with adenovirus (Ad) for red fluorescent protein (RFP, control), TRPC3, TRPC4, TRPC6, or a dominant negative TRPC4 (dnTRPC4) or dnTRPC6. Due to their ability to hetero-oligomerize, the use of a dnTRPC4/6 effectively inhibits the activity of all TRPC subfamilies of channels^{6, 21}(Online Figure II). Ad-RFP infected myocytes showed little or no TRPC-mediated Ca²⁺ entry while myocytes infected with Ad-TRPC3, TRPC4, or TRPC6 showed significant Ca²⁺ entry which was inhibited by expression of Ad-dnTRPC4 or dnTRPC6 but unaffected by nifedipine (Figure 1E). TRPC-mediated Ca²⁺ entry was inhibited by the pan TRPC antagonist SKF-96365 in TRPC3/4/6 infected cells while the more targeted TRPC3/6 inhibitor GSK503A only inhibited TRPC-mediated Ca²⁺ entry in TRPC3/6 infected cells. Similarly, the TRPC3 inhibitor Pyr10³⁷ was able to inhibit TRPC-mediated Ca²⁺ entry in TRPC3 infected cells. Controls were also performed in cells incubated with CPA alone which resulted in a transient Ca²⁺ entry in TRPC4 infected myocytes only but no detectable entry control or TRPC3/6 expressing myocytes (Online Figure ID). This is likely due to the ability of TRPC4 to participate in SOCE while TRPC3/6 tend to be more agonist-induced channels. Similar to our findings in mouse myocytes, TRPC-infected AFMs treated with OAG alone (Online Figure IE) resulted in Ca²⁺ entry and lead to spontaneous contractions.

TRPC channel overexpression in AFMs enhances SR Ca²⁺ in AFMs

TRPC3/6 overexpression in adult mouse heart is linked to cardiac hypertrophy and depressed cardiac contractility^{7, 20}. Myocyte contractions (fractional shortening) and Ca²⁺ transients were measured in AFMs infected with Ad-RFP, -TRPC3, -TRPC4, -TRPC6 and/or –dnTRPC4 or -dnTRPC6 after periods of rest (Figure 2A-C). One of the hallmark contractile characteristics of large mammalian myocytes, including AFMs, is a positive contractile staircase when stimulation is reinstated after a period of rest^{26, 38}. AFMs, as well as those of other large mammals including humans, have a lower cytoplasmic [Na⁺] than found in rodents³⁴. This promotes forward mode Na⁺/Ca²⁺ exchange (NCX) which, in the absence of pacing results in low cytoplasmic [Ca²⁺] and very small amounts of Ca²⁺ stored in the SR. Therefore, in normal AFMs the first post-rest contraction and Ca²⁺ transient are small and then increase in subsequent beats as the SR is progressively loaded with Ca²⁺ to a new a steady state. Following rest periods, control (Ad-RFP) AFMs showed a beat dependent increase in their contractions and Ca²⁺ transients (Figure 2A). Conversely, the first post-rest beat in Ad-TRPC3, -TRPC4, or –TRPC6 infected cells was larger and similar to the steady-state contraction. Intracellular Ca²⁺ was elevated in Ad-TRPC3, -TRPC4, and –TRPC6 infected cells compared to Ad-RFP cells as evidenced by increased fractional shortening (Figure 2A,B) and increased Ca²⁺ transient amplitude (F/F0, Figure 2A,C) in the first paced contraction following a non-paced interval. These TRPC-mediated effects on rested state contractions and Ca²⁺ transients were inhibited by co-expression with dnTRPC4 or dnTRPC6 (Figure 2, Online Figure III). These results suggest that Ca²⁺ influx through TRPC channels maintains SR Ca²⁺ stores in the absence of LTCC mediated Ca²⁺ entry, supporting a role for TRPC-mediated Ca²⁺ entry in disease when the normal pathway for Ca²⁺ influx (through LTCCs) is reduced and the SR Ca²⁺ load is diminished, although the non-diseased adult heart probably does not utilize this pathway as TRPC channels are not appreciably expressed.

Figure 2. TRPC channel overexpression in AFMs enhances SR Ca²⁺ during resting conditions.

A, Representative fractional shortening and Ca²⁺ transient traces from AFMs infected with the indicated adenoviruses and stimulated to pace after a period of rest. Fractional shortening (B) and peak Ca²⁺ transients (C) are represented as the average raw values of the initial beat (left) and as the ratio of the steady state raw values divided by the initial beat raw value (right). p<0.05 was considered significant (ns, p>0.05, *p≤0.05 vs. RFP control; #p≤0.05 vs. raw value of first beat of the same experimental group). All statistical analysis was done on raw values.

Ca²⁺ influx through TRPC channels induces Ca²⁺ spark activity in AFMs

TRPC3/4/6 expressing AFMs had enhanced Ca²⁺ influx at rest that promotes SR Ca²⁺ loading, but steady state contractions were not increased. Therefore, we tested the idea that persistent Ca²⁺ influx through TRPC can lead to excess spontaneous SR Ca²⁺ release (SR Ca²⁺ leak). Spontaneous Ca²⁺ sparks were measured to address this idea³⁹. Ca²⁺ sparks are local spontaneous Ca²⁺ release events caused by the opening of a cluster of ryanodine receptor channels (RyR₂) in the absence of LTCC opening^{40, 41}. These events are common in quiescent rodent myocytes due to their high [Na⁺] that promotes Ca²⁺ entry via reverse mode NCX activity culminating in SR Ca²⁺ overload^{34, 42}. As discussed above, AFMs maintain low [Na²⁺] and they do not exhibit Ca²⁺ accumulation or spontaneous SR Ca²⁺ release in long-term culture³⁴. We infected AFMs with Ad-RFP (control), -TRPC3, -TRPC6, -TRPC3 and -dnTRPC6, or -TRPC6 and dnTRPC4 and measured Ca²⁺ spark activity in the presence and absence of the TRPC channel agonist OAG. Control myocytes rarely exhibited Ca²⁺ sparks but did show a low level of Ca²⁺ spark activity with the addition of OAG (Figure 3A,B). AFMs infected with TRPC3 or TRPC6 showed robust Ca²⁺ spark activity under baseline conditions and this was increased further with OAG stimulation. The majority of detectable Ca²⁺ spark activity was blocked by dnTRPC6, even in the presence of OAG. TRPC3 or TRPC6 mediated Ca²⁺ spark activity was also significantly inhibited by the CaMKII inhibitor KN93 suggesting that this process may in part result from local activation of CaMKII and phosphorylation of RyR₂. To address this issue we measured phosphorylation of RyR at S2814, which is a CaMKII phosphorylation site, in AFMs infected with TRPC3 +/− dnTRPC6 and +/− OAG (Figure 4A-C) or TRPC6 +/− dnTRPC4 and +/− OAG (Online Figure IV). These experiments show that TRPC3 and TRPC6 induce RyR S2814 and phospholamban (PLN) T17 phosphorylation (Figure 4C,F) without modifying RyR S2808 or PLN S16 phosphorylation (Figure 4B,E) or total RyR or PLN expression (Figure 4D,G). OAG mediated increases in RyR S2814 and PLN T17 phosphorylation was reduced by CaMKII inhibition (KN93) (Figure 4C,F, Online Figure IV). We also found that TRPC3 or TRPC6 expression in AFMs was associated with diminished contractile response to catecholamines as evidenced by a reduction in maximal amplitude of fractional shortening and peak Ca²⁺ transients in the presence of isoproterenol (Iso) (Figure 3C, D).

Figure 3. TRPC channels induce SR Ca²⁺ leak and spark activity in AFMs and lead to a reduction in contractility reserve.

A, Representative serial confocal images taken to detect spontaneous Ca²⁺ spark events in AFMs infected with the indicated adenoviruses at baseline or with OAG treatment. Scale bar is 5 μm. Fluorescence intensity of Fluo-4 signal is indicated in a scale of arbitrary units (f.a.u.). KN93 was used at 10umol/L. B, Spark events were quantified and average data of n=20 cells (ns, p>0.05; *** p≤0.001 vs. RFP control; # p≤0.05, ## p≤0.01 vs. TRPC3, & p≤0.05, && p≤0.01 vs. TRPC6). C, Average fractional shortening data and peak Ca²⁺ transients (D) from AFMs infected with the indicated adenovirus at baseline and after exposure to isoproterenol (Iso). p<0.05 was considered significant with * p≤0.05 vs RFP control; #p≤0.05 or ##p≤0.01 vs. baseline of same experimental group.

Figure 4. TRPC channels induce CaMKII mediated RyR₂ and PLN phosphorylation.

Whole cell lysates from AFMs infected with Ad-RFP, -TRPC3, -dnTRPC6, or -TRPC3 and -dnTRPC6 at baseline or treated with OAG (10umol/L) were analyzed by Western with the indicated antibodies. KN93 (10umol/L) was used in addition to OAG where noted. A representative Western panel is shown in A. B-G show average quantified values expressed relative to Ad-RFP control cells at baseline for n=3 experiments. p<0.05 was considered significant (ns, p>0.05, *p≤0.05, **p≤0.01)

TRPC channels localize to caveolae where their organization is essential for hypertrophic signaling

Previous work from our group showed that Ca²⁺ influx through both TRPCs and LTCCs contributes to the activation of Cn-NFAT signaling and indicated that there may be a potential interaction between the two channels²². This interaction might take place within subcellular signaling microdomains such as caveolae²⁵ and it is known that nearly all TRPC isoforms contain a putative caveolin-binding motif⁴³. To explore this further, we characterized the biochemical interactions between TRPCs, LTCCs and caveolin-3 (Cav3), the major structural protein of myocyte caveolae, using immunoprecipitation (IP) with purified plasma membranes from isolated ventricular myocytes of dnTRPC4 transgenic mice (Figure 5A) or AFMs infected with a FLAG-tagged version of TRPC6 (Ad-TRPC6-FLAG, Online Figure VA). Our IP data show that Cav3, LTCCs, and TRPC channels are all complexed together in caveolae. This was further substantiated using sucrose density gradient fractionation of plasma membrane preparations from AFMs infected with Ad-TRPC3 (Figure 5B).

Figure 5. TRPC channels co-localize with LTCCs in caveolae membrane microdomains where their organization is required for hypertrophic signaling.

A, Plasma membranes (PM) were purified from total cell homogenates (H) of isolated myocytes from dnTRPC4 mice. IPs and Westerns were performed with the indicated antibodies (U, unbound fraction; B, bound fraction). B, Sucrose density gradient fractionation on purified PMs from isolated AFMs infected with Ad-TRPC3 confirms the presence of LTCCs and TRPC3 channels in caveolin-3 (Cav3) enriched lipid raft membrane fractions along with the hypertrophic effector calcineurin (Cn) (Fraction 1-Fraction 11, F1-F11). C, AFMs were infected with Ad-NFAT-GFP and the indicated adenoviruses and NFAT translocation was monitored in response to the TRPC agonist OAG (10umol/L) in the presence or absence of MβCD (10mmol/L). Scale bar is 10μm. Average data is represented in D as the nuclear to cytoplasmic GFP ratio of n=100 cells/condition. p<0.05 was considered significant with * p≤0.05; ** p≤0.001; *** p≤0.001 vs. RFP control; ## p≤0.001; ### p≤0.001 vs. TRPC3; && p≤0.001; &&& p≤0.001 vs. TRPC6.

To examine the functional relevance of TRPC channels localized to caveolae, we assessed their role in pathological hypertrophic signaling using an NFAT-GFP reporter assay^{25, 44}. AFMs were infected with Ad-NFAT-GFP and either Ad-RFP (control), -TRPC3, -TRPC4, -TRPC6, -TRPC3 and -dnTRPC6, or -TRPC6 and -dnTRPC4. Essentially all of the NFAT-GFP was localized to the cytoplasm in control AFMs (Figure 5C-D). Ad-TRPC3, -TRPC4, and -TRPC6 infected cells showed a small but significant increase in baseline nuclear NFAT signal, which was inhibited by co-infection with dnTRPC6 or dnTRPC4 (Figure 5C-D, Online Figure VI). Exposing myocytes to OAG caused a very slight increase in NFAT translocation in control cells and a significant increase in nuclear NFAT in TRPC3 or TRPC6 infected cells (Figure 5C,D). Incubating TRPC4 infected cells with high Ca²⁺ (4mmol/L) also lead to a significant increase in NFAT translocation (Online Figure VIC). The TRPC3/4/6 effect was eliminated with co-infection with dnTRPC6 or dnTRPC4 (Figure 5C-D). To further assess whether organizing Ca²⁺ influx pathways in caveolae is essential for NFAT regulation, we subjected myocytes to treatment with methyl-β-cyclodextrin (MβCD), which disrupts caveolae by depleting cholesterol and displaces the macromolecular signaling complexes usually organized in caveolae microdomains (Online Figure V). MβCD inhibited TRPC3/4/6 mediated NFAT nuclear translocation in the presence of OAG or high Ca²⁺ (Figure 5C-D, Online Figure VI), suggesting that the organization of LTCCs and TRPC channels together in caveolae signaling microdomains is necessary for them to activate hypertrophic signaling.

A previous study from our group characterized a caveolae targeted LTCC inhibitor, Rem^1-265-Cav (Rem-Cav), which could specifically inhibit LTCCs with Cav-3 microdomains, to reduce NFAT nuclear translocation without affecting contractility²⁵. When we co-infected NFAT-GFP expressing AFMs with Ad-TRPC3 and -Rem-Cav and treated these cells with OAG we saw a significant inhibition of NFAT translocation (Figure 5C-D). These results suggest that TRPC channels and LTCCs housed together in caveolae membrane microdomains provide a source of Ca²⁺ that induces Cn activation and nuclear NFAT translocation.

TRPCs are relatively nonselective cation channels that allow both Na⁺ and Ca²⁺ entry. Others have shown that Na⁺ entry through the Na/H exchanger (NHE-1) increases local Ca²⁺ via NCX leading to NFAT activation^{45, 46}. Using both Cav3 immunoisolations and sucrose density gradients we found the presence of both NHE-1 and NCX in caveolae membrane microdomains (Online Figure VII). To look specifically at a functional role for NHE-1 in our model, we performed our NFAT-GFP assay in TRPC3/4/6 in the presence of the NHE-1 inhibitor cariporide (Online Figure VID) and found that there was no effect on NFAT translocation in these cells. These data suggest that while NHE-1 co-localizes with TRPC it is not contributing to TRPC-mediated NFAT activation under our conditions.

Loss of TRPC function protects against cardiac dysfunction progression after MI and improves survival

After MI, myocytes develop pathological hypertrophy, myocyte function is altered and TRPC channel expression increases. Overexpression of TRPC channels in the mouse heart is sufficient to induce hypertrophy and cardiomyopathy^{5, 7, 20} and mice expressing dominant negative versions of the channel have less hypertrophy in response to pressure-overload or neuroendocrine agonist infusion^{8, 21} Taken together with our in vitro findings these data support the idea that inhibiting TRPC function in the heart could be beneficial after MI. To test this idea we used a transgenic mouse with cardiac-specific expression of a truncated dominant negative TRPC4 (dnTRPC4)²¹ that reduces the activity of both the TRPC1/4/5 and TRPC3/6/7 subfamilies of TRPC channels²¹. TRPC mRNA levels were not significantly different between WT and dnTRPC4 mice both in sham and MI groups (Figure 6 and Online Figure VIII) with the exception of TRPC4 which was markedly increased in dnTRPC4 animals due to over-expression of the transgene. TRPC-mediated Ca²⁺ entry seen in WT 6-week MI myocytes, which could be partially inhibited by the selective TRPC3/6 antagonist GSK503A or the TRPC3 inhibitors Pyr3 or Pyr10 or completely inhibited with SKF-96365, was not present in dnTRPC4 mice (Figure 6). ECHO measurements revealed a slightly increased baseline ejection fraction (EF) in dnTRPC4 mice compared to wild-type (WT) animals (75.5 vs 68.2%) (Figure 7A-B), consistent with the inotropic effects of dnTRPC6 observed in AFMs. The area at risk (AAR) after MI was identical in WT and dnTRPC4 mice (41.3±3.8% vs. 43.5±4.9%; Online Figure IIIA). Infarct length measured 3 weeks after MI was not significantly different in dnTRPC4 than in WT mice (dnTRPC4 vs. WT: 28.7±1.7% vs. 33.2±0.9%) (Online Figure IXB). In addition, the dilation seen in WT mice 3 weeks post-MI was attenuated in dnTRPC4 hearts (Online Figure IXC,D).

Figure 6. TRPC channel expression and activity in WT and dnTRPC4 TG mice.

A, RT-PCR shows an up-regulation of TRPC1/3/4/6 channel isoforms 6-weeks post-MI along with the activation of the fetal gene program compared to sham animals. dnTRPC4 TG myocytes show similar levels of baseline TRPC expression levels and TRPC1/3/6 up-regulation post-MI but show reduced ANF and SMA compared to WT mice post-MI. B, TRPC-mediated Ca²⁺ entry in isolated myocytes from sham mice (left) dnTRPC4 TG mice (right) post-MI in the presence of the TRPC channel agonist OAG (10umol/L) and the SERCA inhibitor CPA (5umol/L). Where indicated, the TRPC antagonists SKF-96365 (5umol/L), GSK503A (GSK, 10umol/L), Pyr3 (3umol/L), Pyr10 (3umol/L) or the LTCC inhibitor nifedipine (Nif, 10umol/L) were used. p<0.05 was considered significant (ns, p>0.05; *p≤0.05, **p≤0.01, ***p≤0.001 vs. WT sham; #p≤0.05, ##p≤0.01, ; ###p≤0.001 vs. dnTRPC4 sham; &p≤0.05 vs. WT 6wk MI).

Figure 7. Cardiac function and survival was improved and pathological remodeling attenuated in dnTRPC4 mice vs WT animals post-MI.

A, Representative M-mode tracings from sham and MI animals at 6-weeks post-MI. Average cardiac ejection fraction (B), posterior wall thickness (C), and LV internal diameter (D) were measured by Echocardiography in sham and MI mice at baseline and 2 and 6 weeks post-MI. E, 6-week survival data analyzed using a Kaplan-Meier regression of WT vs dnTRPC4 mice. Significance was determined using the long-rank test. Heart weight (HW, F) or lung weight (LungW, G) normalized to BW measured in sham and MI mice after 6-weeks. p<0.05 was considered significant (ns, p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001).

Serial ECHO was used to measure LV structure and function after MI. WT (EF, sham vs MI: 68.2 vs 37.2%) and dnTRPC4 (EF, sham vs MI: 75.5 vs 41.8%) animals had equivalent reductions in cardiac pump function 2 weeks after MI (Figure 7B). LV function remained depressed at 6-weeks post-MI in WT animals while there was a significant improvement in cardiac pump function in dnTRPC4 hearts 6 weeks after MI (Figure 7A,B).

There were significant pathological changes in ventricular geometry and wall thickness in all hearts after MI (Figure 7C-D). The magnitude of these changes increased with time in WT animals but was attenuated in dnTRPC4 mice. After MI, posterior wall thickness (PWT) was decreased in both WT and dnTRPC4 hearts (WT pre-MI vs. post-MI: 0.97 vs. 0.87mm; dnTRPC4: 1.06 vs. 0.84mm) (Figure 7C). At 6 weeks post-MI, PWT returned to values near pre-MI levels (or close to shams) in dnTRPC4 hearts while PWT remained thinner in WT hearts. All hearts showed some evidence of dilation after MI, however, LV internal diameter (LVID) increased significantly more in WT than in dnTRPC4 hearts in the first 2 weeks after MI (WT pre-MI vs. post-MI: 3.8 vs. 4.9 mm; dnTRPC4: 3.7 vs. 4.5 mm) (Figure 7D). By 6 weeks post-MI, LVID was significantly more dilated in WT hearts than in dnTRPC4 (WT vs. dnTRPC4: 5.3 vs. 4.8 mm).

dnTRPC4 mice had significantly greater survival after MI (53.7%) than WT animals (27.5%) during the 6-week post-MI study period (Figure 7E). Heart Weight/Body Weight (HW/BW) and Lung Weight/Body Weight (LungW/BW) were significantly increased (Figure 7F,G) in WT versus dnTRPC4 mice 6 weeks post-MI. There were minimal changes in liver weight/body weight ratio before and after MI, and there was no significant difference between dnTRPC4 and WT mice (Online Figure IXE). Dephosphorylation of NFAT induces cytoplasmic to nuclear translocation. 6-week MI dnTRPC4 mice had increased levels of phosphorylated NFAT as compared to WT 6-week MI mice indicating reduced NFAT dephosphorylation in the dnTRPC4 mice (Online Figure XA-C). In accordance with this, isolated myocytes from 2- and 6-week post-MI WT had significantly greater length and width than dnTRPC4 mice (Online Figure XD,E). Collectively these results show that dnTRPC4 animals have less pathological remodeling after MI, improved cardiac pump function and enhanced survival.

Myocytes from dnTRPC4 mice retain their hypercontractile phenotype after MI

One potential mechanism for the improved cardiac function after MI in dnTRPC4 versus WT hearts is that myocyte function and adrenergic responsiveness are better preserved. To address this idea we first measured twitch contractions and [Ca²⁺]_i transients in the absence and presence of Iso in dnTRPC4 and WT myocytes after sham or MI procedures. Representative data are shown in Online Figure XIA,B. Fractional shortening (FS) of dnTRPC4 was significantly greater than in WT (sham) myocytes (dnTRPC4 vs. WT: 11.0±0.6% vs. 8.4±0.6%) (Figure 8A). After MI, contractions remained significantly greater in dnTRPC4 than in WT myocytes (dnTRPC4 vs. WT: 13.4±1.1% vs. 8.9±0.4%).

Figure 8. Fractional shortening, Ca²⁺ transients, and L-type Ca²⁺ current (I_Ca,L) measured in isolated cardiac myocytes from sham and post-MI hearts.

Cellular fractional shortening, Ca²⁺ transients and I_Ca,L were measured in myocytes isolated from sham and 3-week post-MI WT and dnTRPC4 mice in the presence and absence of isoproterenol (Iso). Average data are shown for fractional shortening (A), peak Ca²⁺ transients (B), fractional shortening half width (C), Ca²⁺ decay rate (Tau,D), peak I_Ca,L (E), and membrane capacitance (F). p<0.05 was considered significant (ns, p>0.05, *p≤0.05, **p≤0.01)

Myocyte contractions in both WT and dnTRPC4 cells increased with Iso (WT sham −/+ Iso: 8.4±0.6% vs. 11.7±0.8%; dnTRPC4 sham −/+ Iso: 11.0±0.6% vs. 13.5±0.6%) with baseline contractions in dnTRPC4 being greater than in WT (Figure 8A). Post-MI, dnTRPC4 myocytes again had greater baseline contractions and Iso response than WT myocytes (post-MI WT −/+ Iso: 8.9±0.4% vs. 13.0±0.4%; post-MI dnTRPC4 −/+ Iso: 13.4±1.1% vs. 16.0±0.8%).

Peak systolic [Ca²⁺]_i in dnTRPC4 (sham) myocytes was significantly greater than in WT myocytes (Figure 8B), explaining their greater twitch contractions. After MI, [Ca²⁺]_i transients in dnTRPC4 myocytes remained significantly greater than in WT myocytes (peak F/F₀ in dnTRPC4 sham vs. WT sham: 3.0±0.3 vs. 2.1±0.3; post-MI dnTRPC4 vs. WT: 3.6±0.5 vs. 2.5±0.2). Iso significantly increased Ca²⁺ transient amplitude in both WT and dnTRPC4 myocytes, with Ca²⁺ transient amplitude being significantly greater in dnTRPC4 myocytes (WT (sham) −/+ Iso: 2.1±0.3 vs. 3.5±0.4; dnTRPC4 (sham) −/+ Iso: 3.0±0.3 vs. 4.4±0.5; post-MI WT −/+ Iso: 2.5±0.2 vs. 3.6±0.6; post-MI dnTRPC4 −/+ Iso: 3.6±0.5 vs. 6.0±0.8) (Figure 8B).

Contraction half width and the time constant of decay (Tau) of [Ca²⁺]_i transients were also measured. Half width (−/+ Iso conditions) was significantly less in dnTRPC4 (sham) versus WT (sham) myocytes (dnTRPC4 (sham) −/+ Iso: 220±11 vs. 209±9ms; WT (sham) −/+ Iso: 280±20 vs. 261±17ms) (Figure 8C). MI induced changes (half width increase) in the duration of contractions in both dnTRPC4 and WT myocytes. After MI, half width of contractions (−/+ Iso conditions) remained significantly less in dnTRPC4 versus WT myocytes (dnTRPC4 (MI) −/+ Iso: 247±16 vs. 230±14ms; WT (MI) −/+ Iso: 315±15 vs. 279±11ms) (Figure 8C). Iso induced significant decreases in Tau in both WT and dnTRPC4 myocytes after sham or MI (Figure 8D). There were no significant differences in Tau between WT and dnTRPC4 myocytes after sham or MI −/+ Iso conditions (Figure 8D). Collectively, these data show that myocytes from dnTRPC4 MI hearts retain a hypercontractile phenotype after MI.

LTCC current (I_Ca,L) was not different between dnTRPC4 and WT myocytes after sham or MI

Altered function of the LTCC and loss of adrenergic regulation is a common feature of diseased cardiac myocytes⁴⁷. We next examined if loss of TRPC function influenced the behavior of LTCCs either before or after MI. LTCC currents were measured in single isolated myocytes from WT and dnTRPC4 hearts with or without MI. I_Ca,L density was not significantly different in sham dnTRPC4 versus WT myocytes (peak I_Ca,L in dnTRPC4 vs. WT: −13.0±1.4 vs. −12.1±0.85pA/pF) (Figure 8E). Iso increased I_Ca,L density in both sham dnTRPC4 myocytes (pre-Iso vs. after Iso: −13.0 ±1.4 vs. −22.5±2.8 pA/pF) and sham WT myocytes (−12.1±0.85 to −20.9± 1.9 pA/pF). After MI, I_Ca,L density was decreased to a similar extent in all myocytes (dnTRPC4 vs. WT: −11.8 ±1.7 vs. −10.4 ±1.1 pA/pF)( Figure 8E). However, only dnTRPC4 myocytes showed a significant increase in I_Ca,L with Iso after MI (pre-Iso vs. after Iso: −11.8±1.7 vs. −18.3±2.5 pA/pF). Cell capacitance (Figure 8F) and cell size (Online Figure XD,E) were similar in sham WT and dnTRPC4 animals but a significant increase in cell capacitance was seen in WT cells following MI (Figure 8F) but not in dnTRPC4 myocytes.

DISCUSSION

This study explored the idea that Ca²⁺ influx through TRPC channels expressed after MI contributes to altered myocyte contractility and hypertrophic signaling. Our studies revealed a low level of TRPC isoform expression in normal adult mouse and adult feline ventricular myocytes (AFMs), with a significant increase in select TRPC isoform expression after MI (Figure 1). TRPC was shown to induce SOCE, which was abolished with a dnTRPC6 expressing adenovirus. Using AFMs we showed that Ca²⁺ entry through TRPC3 could load the SR when SR Ca²⁺ stores were naturally depleted but could overload the SR and cause spontaneous SR Ca²⁺ release (Ca²⁺ sparks, leak) if SOCE was persistent or excessive, and these effects appear to be due to CaMKII mediated RyR S2814 phosphorylation (Figure 4). TRPC expression was associated with reduced contractile effects of catecholamines. Our studies also showed that a fraction of TRPC channels are localized to caveolae, where together with LTCCs, they activate pathological hypertrophic signaling. Finally, mice with cardiac myocyte specific expression of dnTRPC4 had less cardiac dysfunction and adverse remodeling after MI.

TRPC expression in disease

A clear link between Ca²⁺ influx and cardiac hypertrophy has been established^{1, 48} and activation of Cn-NFAT signaling is known to initiate the coordinated expression of maladaptive hypertrophic genes, and over stimulation of this pathway can lead to heart failure^{3, 49, 50}. Multiple in vitro^{5, 22, 24, 51, 52} and in vivo^{7, 20, 21, 51, 53} TRPC expression systems have documented a role for these channels in the induction of Cn-NFAT signaling and subsequent hypertrophic remodeling. TRPC loss-of-function^{6, 8, 21, 24, 54} and selective inhibition^{23, 24, 53} animal models are protected against cardiac hypertrophy and indices of heart failure following either pressure overload or neurohormonal stress. In accordance with these studies, we found that cardiac specific overexpression of dnTRPC4 resulted in reduced pathological remodeling in an MI model of injury (Figure 7) and a cardioprotective phenotype that increased survival post-MI.

TRPC, SOCE, and myocyte contractility

Progressive deterioration of cardiac contractility is a central feature of heart failure and alterations of intracellular Ca²⁺ regulation are primarily responsible for this depression in contractility reserve^{1, 47}. In this study we explored the hypothesis that TRPC channels expressed in diseased myocytes contribute to their deteriorating contractility. Others have found that cardiac specific overexpression of TRPC6 in mice resulted in an exaggerated hypertrophic response to pressure overload with decreased systolic function⁷ and a similar study showed that TRPC3 TG mice developed a loss of ventricular performance with profound cardiomyopathy²⁰. We found enhanced cardiac pump function in cardiac specific dnTRPC4 mice (Figure 7A-B) and increased myocyte fractional shortening and peak Ca²⁺ transients (Figure 8A-B). These are somewhat curious findings, since TRPC is a pathway for Ca²⁺ influx and this would be expected to increase rather than decrease contractile Ca²⁺. To examine if/how TRPC channels cause alterations in myocyte contractility, TRPC3, TRPC4, TRPC6 and/or dnTRPC6 and dnTRPC4 were expressed in AFMs. We found that the changes in AFM contractile function were dependent on the experimental conditions. When normal or RFP infected AFMs were unpaced for a period of time, their SR Ca²⁺ stores became depleted (Figure 2A-C). TRPC3/4/6 expression resulted in enhanced rested state contractions (Figure 2A-C), suggesting that when SR Ca²⁺ stores are depleted, TRPC channels can supply Ca²⁺ for refilling. However, in paced AFMs, TRPC3/6 resulted in reduced steady state contractile function and reduced responsiveness to catecholamines (Figure 3C). TRPC3/6 overexpression induced Ca²⁺ sparks (Figure 3A,B), suggesting that altered contractility was due to enhanced SR Ca²⁺ leak. Finally we showed that increased TRPC3/6 activity was associated with RyR S2814 phosphorylation that was reduced by CaMKII inhibition (Figure 4A,C). These results suggest that while Ca²⁺ influx through TRPC channels can replenish depleted SR Ca²⁺ stores, excess TRPC channel activity causes local activation of CaMKII and phosphorylation of RyR at S2814 resulting in abnormal RyR function, producing spontaneous diastolic SR Ca²⁺ release leading to depressed contractility reserve.

TRPC and hypertrophy signaling

Many groups have shown that TRPC channels contribute to the activation of Cn-NFAT signaling and the hypertrophic response^{6, 21, 22, 55}. Data from our study is in accordance with this and expands upon what is known to show that the organization of TRPC channels along with LTCCs in caveolae membrane microdomains influences their ability to orchestrate Cn-NFAT signaling. TRPC channels also allow both Na⁺ and Ca²⁺ influx and it has been shown by others that Na⁺ entry via the Na/H exchanger (NHE-1) and induce Ca²⁺ entry via the NCX to activate NFAT activation^{45, 46, 60}. We found both NHE-1 and NCX in caveolae membrane microdomains (Online Figure VII) suggesting that they could contribute to TRPC-mediated NFAT activation in these signaling microdomains. We addressed this by inhibiting NHE-1 with cariporide (Online Figure VID) but saw no change in TRPC mediated NFAT nuclear translocation, indicating NHE-1 is not playing a central role in this process in our system. However, our experimental design is unable to definitively rule out a role for Na⁺ in this process and it is possible that TRPC-mediated Na⁺ entry is also a contributing factor to Cn-NFAT.

TRPC inhibition post-MI

Finally, an in depth characterization of an MI model of injury in dnTRPC4 TG mice was used to determine if reducing TRPC channel activity after MI reduced structural and functional remodeling and had a beneficial outcome. We found that dnTRPC4 mice did not exhibit TRPC-mediated Ca²⁺ entry after MI and had less pathological hypertrophy, better cardiac performance, less progression of heart failure, and increased survival after MI compared to WT animals (Figures 6-7, Online Figures IX, X).

Collectively, our studies show that TRPC channels are stress response molecules that are up regulated in chronic cardiac disease states. Mechanistically, our data suggests that TRPC channels disrupt normal SR Ca²⁺ storage by inducing SR Ca²⁺ leak to contribute to depressed contractility reserve in disease. These effects are accompanied by coordinated Ca²⁺ activated Cn-NFAT signaling through caveolae membrane microdomains. These data suggest that targeted inhibition of cardiac myocyte TRPC channels might be an effective strategy for attenuating pathological structural remodeling and for maintaining contractility reserve after MI.

Novelty and Significance.

What Is Known?

• Transient receptor potential (canonical) (TRPC) channels are present in low abundance in the normal heart and their expression is increased by pressure overload.

• TRPC channels contribute to cardiac hypertrophy and disease progression in pressure overload.

What New Information Does This Article Contribute?

• TRPC channel expression increases after myocardial infarction (MI) and is associated with pathological remodeling and contractility reserve defects, inhibition of TRPC channels post-MI increased survival, reduced pathological remodeling, and improved cardiac function.

• Ca²⁺ influx through TRPC channels activates Ca²⁺/calmodulin protein kinase II (CaMKII) resulting in Ca²⁺ leak from sarcoplasmic reticulum stores and reduces contractility reserve.

• TRPC channels organized in caveolae membrane signaling microdomains provide a local Ca²⁺ signal that activates Calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling, a well-established upstream mediator of pathological hypertrophy.

TRPC channel expression and activity are increased in models of pathological hypertrophy and heart failure. We asked if and how Ca²⁺ influx through TRPC channels contributes to the structural remodeling and contractility defects seen after MI. We found that isoforms of the TRPC channel were upregulated after MI. The biological activity of TRPC channels was linked to reduced SR Ca²⁺ stores using an in vitro system. Our studies showed that TRPC activity triggered spontaneous SR Ca²⁺ release that was linked to CaMKII activation and downstream modification of ryanodine receptors making them more prone to leak. These changes resulted in a reduction of contractility reserve. Our results showed that TRPC channels localized to caveolae membrane domains are involved in stress mediated activation of Cn-NFAT signaling. TRPC channel inhibition with a cardiac specific dominant negative TRPC construct reduced pathological structural and functional remodeling after MI and improved survival. These studies suggest that following MI, the biological activity of TRPC channels perpetuates cardiac hypertrophy and contributes to depression of contractility reserve.

Nonstandard Abbreviations and Acronyms

AFMs

adult feline myocytes

Cav

caveolin

Cn

calcineurin

ECHO

echocardiography

Iso

isoproterenol

LTCC

L-type Ca²⁺ channel

MβCD

methyl-β-cyclodextrin

MI

myocardial infarction

NCX

Na⁺/ Ca²⁺ exchanger

NFAT

nuclear factor of activated T cells

NHE

Na⁺/ H⁺ exchanger

OAG

1-oleoyl-2-acetyl-sn-glycerol

PLN

phospholamban

RFP

red fluorescent protein

RyR

ryanodine receptor

SERCA

sarcoplasmic reticulum Ca²⁺ ATPase

SOCE

store operated Ca²⁺ entry

SR

sarcoplasmic reticulum

TG

transgenic

TRPC

transient receptor potential (canonical)