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 reverse transcriptase polymerase chain reaction. 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 with sham animals (Figure 1A).

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Figure 1.

Myocardial infarction induces transient receptor potential canonical (TRPC) channel expression and activity in mice, and overexpression of TRPC channels in feline myocytes leads to increased membrane Ca²⁺ influx. A, Reverse transcriptase polymerase chain reaction shows an upregulation of TRPC1/3/4/6 channel isoforms at 1, 2, and 6 wks post-myocardial infarction (MI) along with the activation of the fetal gene program. B to D, TRPC-mediated Ca²⁺ entry in isolated myocytes from sham mice (left) or 1 wk (B), 2 wk (C), or 6 wk (D) post-MI mice (right) in the presence of the TRPC channel agonist OAG (10 umol/L) and the sarcoplasmic reticulum Ca²⁺ ATPase inhibitor cyclopiazonic acid (CPA; 5 umol/L). Where indicated, the TRPC antagonists SKF-96365 (5 umol/L), GSK503A (GSK; 10 umol/L), Pyr10 (3 umol/L), or the l-type Ca²⁺ channel inhibitor nifedipine (Nif; 10 umol/L) were used. E, Adult feline myocytes 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). ANF indicates atrial natriuretic factor; BNP, brain natriuretic peptide; MHC, myosin heavy chain; and SMA, skeletal muscle actin.

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 SOCE through defined channel complexes that include STIM (stromal interaction molecule) and Orai and potentially TRPC channels on the plasma membrane.³² Although the role of TRPC channels in SOCE in cardiac myocytes is not well defined, sarcoplasmic reticulum (SR) Ca²⁺ depletion of isolated myocytes followed by the reintroduction 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, whereas myocytes from sham animals showed no detectable activity (Figure 1B–1D). 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–1D). 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 cyclopiazonic acid 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 led to spontaneous contractions.

AFMs Expressing TRPC Channels Have Increased Membrane Ca²⁺ Influx

To further characterize the properties of TRPC channels in adult cardiac myocytes, we used cultured isolated 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 electric 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 dnTRPC4 or dnTRPC6. Because of 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, whereas myocytes infected with Ad-TRPC3, TRPC4, or TRPC6 showed significant Ca²⁺ entry, which was inhibited by the 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, whereas 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 cyclopiazonic acid alone, which resulted in a transient Ca²⁺ entry in TRPC4-infected myocytes only but no detectable entry in control or TRPC3/6 expressing myocytes (Online Figure ID). This is likely because of the ability of TRPC4 to participate in SOCE, whereas TRPC3/6 tends 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 led to spontaneous contractions.

TRPC Channel Overexpression in AFMs Enhances SR Ca²⁺ in AFMs

TRPC3/6 overexpression in the 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 or -dnTRPC4 or -dnTRPC6 after periods of rest (Figure 2A–2C). 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 rodents.³⁴ This promotes forward-mode Na⁺/Ca²⁺ exchange (NCX), which in the absence of pacing results in low cytoplasmic [Ca]²⁺ 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 steady state. After 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 with Ad-RFP cells as evidenced by increased fractional shortening (Figure 2A and 2B) and increased Ca²⁺ transient amplitude (F/F0; Figure 2A and 2C) in the first paced contraction after a nonpaced interval. These TRPC-mediated effects on rested state contractions and Ca²⁺ transients were inhibited by coexpression 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 LTCC) is reduced and the SR Ca²⁺ load is diminished, although the nondiseased adult heart probably does not use this pathway because TRPC channels are not appreciably expressed.

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Figure 2.

Transient receptor potential canonical channel overexpression in adult feline myocytes (AFMs) enhances sarcoplasmic reticulum 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 red fluorescent protein (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 idea.³⁹ Ca²⁺ sparks are local spontaneous Ca²⁺ release events caused by the opening of a cluster of ryanodine receptor (RyR) channels in the absence of LTCC opening.^40,41 These events are common in quiescent rodent myocytes because of 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 [Na]²⁺, and they do not exhibit Ca²⁺ accumulation or spontaneous SR Ca²⁺ release in long-term culture.³⁴ 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 and 3B). 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 Ca²⁺/calmodulin protein kinase II (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–4C) or TRPC6±dnTRPC4 and ±OAG (Online Figure IV). These experiments show that TRPC3 and TRPC6 induce RyR S2814 and phospholamban T17 phosphorylation (Figure 4C and 4F) without modifying RyR S2808 or phospholamban S16 phosphorylation (Figure 4B and 4E) or total RyR or phospholamban expression (Figure 4D and 4G). OAG-mediated increases in RyR S2814 and phospholamban T17 phosphorylation was reduced by CaMKII inhibition (KN93; Figure 4C and 4F; 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 (Figure 3C and 3D).

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Figure 3.

Transient receptor potential canonical (TRPC) channels induce sarcoplasmic reticulum Ca²⁺ leak and spark activity in adult feline myocytes (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 10 umol/L. B, Spark events were quantified and average data of n=20 cells (ns, P>0.05; ***P≤0.001 vs red fluorescent protein [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.

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Figure 4.

Transient receptor potential canonical (TRPC) channels induce Ca²⁺/calmodulin protein kinase II–mediated ryanodine receptor 2 (RyR2) and phospholamban (PLN) phosphorylation. Whole-cell lysates from adult feline myocytes infected with Ad-RFP, -TRPC3, -dnTRPC6, or -TRPC3 and -dnTRPC6 at baseline or treated with OAG (10 umol/L) were analyzed by Western blot with the indicated antibodies. KN93 (10 umol/L) was used in addition to OAG where noted. A representative Western blot is shown in A. B to G, 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 LTCC contributes to the activation of Cn-NFAT signaling and indicated that there may be a potential interaction between the channels.²² This interaction might take place within subcellular signaling microdomains such as caveolae,²⁵ and it is known that nearly all TRPC isoforms contain a putative caveolin-binding motif.⁴³ To explore this further, we characterized the biochemical interactions between TRPCs, LTCC, and caveolin-3, the major structural protein of myocyte caveolae, using immunoprecipitation 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 immunoprecipitation data show that caveolin-3, LTCC, 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).

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Figure 5.

Transient receptor potential canonical (TRPC) channels colocalize with l-type Ca²⁺ channel (LTCC) in caveolae membrane microdomains where their organization is required for hypertrophic signaling. A, Plasma membranes (PMs) were purified from total cell homogenates (H) of isolated myocytes from dominant-negative TRPC4 mice. Immunoprecipitations (IPs) and Westerns were performed with the indicated antibodies (B indicates bound fraction; and U, unbound fraction). B, Sucrose density gradient fractionation on purified PMs from isolated adult feline myocytes (AFMs) infected with Ad-TRPC3 confirms the presence of LTCC and TRPC3 channels in caveolin-3 (Cav3)–enriched lipid raft membrane fractions along with the hypertrophic effector calcineurin (Cn; fraction 1 to 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 (10 umol/L) in the presence or absence of methyl-β-cyclodextrin (MβCD; 10 mmol/L). Scale bar is 10 μm. Average data are represented in D as the nuclear to cytoplasmic GFP ratio of n=100 cells per condition. P<0.05 was considered significant with *P≤0.05; **P≤0.001; ***P≤0.001 vs red fluorescent protein 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 and 5D). Ad-TRPC3, -TRPC4, and -TRPC6–infected cells showed a small but significant increase in baseline nuclear NFAT signal, which was inhibited by coinfection with dnTRPC6 or dnTRPC4 (Figure 5C and 5D; 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 and 5D). Incubating TRPC4-infected cells with high Ca²⁺ (4 mmol/L) also led to a significant increase in NFAT translocation (Online Figure VIC). The TRPC3/4/6 effect was eliminated with coinfection with dnTRPC6 or dnTRPC4 (Figure 5C and 5D). To further assess whether organizing Ca²⁺ influx pathways in caveolae is essential for NFAT regulation, we subjected myocytes to treatment with methyl-β-cyclodextrin, which disrupts caveolae by depleting cholesterol and displaces the macromolecular signaling complexes usually organized in caveolae microdomains (Online Figure V). Methyl-β-cyclodextrin inhibited TRPC3/4/6-mediated NFAT nuclear translocation in the presence of OAG or high Ca²⁺ (Figure 5C and 5D; Online Figure VI), suggesting that the organization of LTCC 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, which could specifically inhibit LTCC with caveolin-3 microdomains, to reduce NFAT nuclear translocation without affecting contractility.²⁵ When we coinfected NFAT-GFP–expressing AFMs with Ad-TRPC3 and -Rem^1-265-Cav and treated these cells with OAG, we saw a significant inhibition of NFAT translocation (Figure 5C and 5D). These results suggest that TRPC channels and LTCC housed together in caveolae membrane microdomains provide a source of Ca²⁺ that induces calcineurin 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 caveolin-3 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 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 although NHE-1 colocalizes 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 dnTRPC4²¹ that reduces the activity of both the TRPC1/4/5 and TRPC3/6/7 subfamilies of TRPC channels.²¹ TRPC mRNA levels were not significantly different between wild-type (WT) and dnTRPC4 mice both in sham and MI groups (Figure 6; Online Figure VIII) with the exception of TRPC4, which was markedly increased in dnTRPC4 animals because of overexpression 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). Echocardiography measurements revealed a slightly increased baseline ejection fraction in dnTRPC4 mice compared with WT animals (75.5% versus 68.2%; Figure 7A and 7B), consistent with the inotropic effects of dnTRPC6 observed in AFMs. The area at risk after MI was identical in WT and dnTRPC4 mice (41.3±3.8% versus 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 versus WT: 28.7±1.7% versus 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 andn IXD).

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Figure 6.

Transient receptor potential canonical (TRPC) channel expression and activity in wild-type (WT) and dominant-negative (dn) TRPC4 transgenic (TG) mice. A, Reverse transcriptase polymerase chain reaction shows an upregulation of TRPC1/3/4/6 channel isoforms 6 wk post–myocardial infarction (MI) along with the activation of the fetal gene program compared with sham animals. dnTRPC4 TG myocytes show similar levels of baseline TRPC expression levels and TRPC1/3/6 upregulation post-MI but show reduced atrial natriuretic factor (ANF) and skeletal muscle actin (SMA) compared with WT mice post-MI. B, TRPC-mediated Ca²⁺ entry in isolated myocytes from sham mice (left) and dnTRPC4 TG mice (right) post-MI in the presence of the TRPC channel agonist OAG (10 umol/L) and the sarcoplasmic reticulum Ca²⁺ ATPase inhibitor cyclopiazonic acid (5 umol/L). Where indicated, the TRPC antagonists SKF-96365 (5 umol/L), GSK503A (GSK; 10 umol/L), Pyr3 (3 umol/L), Pyr10 (3 umol/L) or the l-type Ca²⁺ channel inhibitor nifedipine (Nif; 10 umol/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 6 wk MI). BNP indicates brain natriuretic peptide; and MHC, myosin heavy chain.

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Figure 7.

Cardiac function and survival was improved and pathological remodeling attenuated in dominant-negative transient receptor potential canonical isoform 4 (dnTRPC4) mice vs wild-type (WT) animals post–myocardial infarction (MI). A, Representative M-mode tracings from sham and MI animals at 6 wk post-MI. Average cardiac ejection fraction (B), posterior wall thickness (C), and left ventricular (LV) internal diameter (D) were measured by echocardiography in sham and MI mice at baseline and 2 and 6 wk post-MI. E, Six-week survival data analyzed using a Kaplan–Meier regression of WT vs dnTRPC4 mice. Significance was determined using the log-rank test. Heart weight (HW; F) or lung weight (LungW; G) normalized to body weight (BW) measured in sham and MI mice after 6 wks. P<0.05 was considered significant (ns, P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001).

Serial echocardiography was used to measure left ventricular structure and function after MI. WT (ejection fraction: sham versus MI, 68.2% versus 37.2%) and dnTRPC4 (ejection fraction: sham versus MI, 75.5% versus 41.8%) animals had equivalent reductions in cardiac pump function 2 weeks after MI (Figure 7B). Left ventricular function remained depressed at 6 weeks post-MI in WT animals, whereas there was a significant improvement in cardiac pump function in dnTRPC4 hearts 6 weeks after MI (Figure 7A and 7B).

There were significant pathological changes in ventricular geometry and wall thickness in all hearts after MI (Figure 7C and 7D). The magnitude of these changes increased with time in WT animals but was attenuated in dnTRPC4 mice. After MI, posterior wall thickness was decreased in both WT and dnTRPC4 hearts (WT pre-MI versus post-MI: 0.97 versus 0.87 mm; dnTRPC4: 1.06 versus 0.84 mm; Figure 7C). At 6 weeks post-MI, posterior wall thickness returned to values near pre-MI levels (or close to shams) in dnTRPC4 hearts, whereas posterior wall thickness remained thinner in WT hearts. All hearts showed some evidence of dilation after MI; however, left ventricular internal diameter increased significantly more in WT than in dnTRPC4 hearts in the first 2 weeks after MI (WT pre-MI versus post-MI: 3.8 versus 4.9 mm; dnTRPC4: 3.7 versus 4.5 mm; Figure 7D). By 6 weeks post-MI, left ventricular internal diameter was significantly more dilated in WT hearts than in dnTRPC4 (WT versus dnTRPC4: 5.3 versus 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 and lung weight/body weight were significantly increased (Figure 7F and 7G) 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. Six-week MI dnTRPC4 mice had increased levels of phosphorylated NFAT as compared with WT 6-week MI mice, indicating reduced NFAT dephosphorylation in dnTRPC4 mice (Online Figure XA–XC). In accordance with this, isolated myocytes from 2- and 6-week post-MI WT had significantly greater length and width than in dnTRPC4 mice (Online Figure XD and XE). 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 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 [Ca]²⁺_i transients in the absence and presence of isoproterenol in dnTRPC4 and WT myocytes after sham or MI procedures. Representative data are shown in Online Figure XIA and XIB. Fractional shortening of dnTRPC4 was significantly greater than in WT (sham) myocytes (dnTRPC4 versus WT: 11.0±0.6% versus 8.4±0.6%; Figure 8A). After MI, contractions remained significantly greater in dnTRPC4 than in WT myocytes (dnTRPC4 versus WT: 13.4±1.1% versus 8.9±0.4%).

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Figure 8.

Fractional shortening (FS), Ca²⁺ transients, and l-type Ca²⁺ current (I_Ca,L) measured in isolated cardiac myocytes from sham and post–myocardial infarction (MI) hearts. Cellular FS, Ca²⁺ transients, and I_Ca,L were measured in myocytes isolated from sham and 3-wk post-MI wild-type and dominant-negative transient receptor potential canonical isoform 4 mice in the presence and absence of isoproterenol (Iso). Average data are shown for FS (A), peak Ca²⁺ transients (B), FS 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 isoproterenol (WT sham±isoproterenol: 8.4±0.6% versus 11.7±0.8%; dnTRPC4 sham±isoproterenol: 11.0±0.6% versus 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 isoproterenol response than WT myocytes (post-MI WT±isoproterenol: 8.9±0.4% versus 13.0±0.4%; post-MI dnTRPC4±isoproterenol: 13.4±1.1% versus 16.0±0.8%).

Peak systolic [Ca]²⁺_i in dnTRPC4 (sham) myocytes was significantly greater than in WT myocytes (Figure 8B), explaining their greater twitch contractions. After MI, [Ca]²⁺_i transients in dnTRPC4 myocytes remained significantly greater than in WT myocytes (peak F/F₀ in dnTRPC4 sham versus WT sham: 3.0±0.3 versus 2.1±0.3; post-MI dnTRPC4 versus WT: 3.6±0.5 versus 2.5±0.2). Isoproterenol significantly increased Ca²⁺ transient amplitude in both WT and dnTRPC4 myocytes, with Ca²⁺ transient amplitude being significantly greater in dnTRPC4 myocytes (WT [sham]±isoproterenol: 2.1±0.3 versus 3.5±0.4; dnTRPC4 [sham]±isoproterenol: 3.0±0.3 versus 4.4±0.5; post-MI WT±isoproterenol: 2.5±0.2 versus 3.6±0.6; post-MI dnTRPC4±isoproterenol: 3.6±0.5 versus 6.0±0.8; Figure 8B).

Contraction half width and the time constant of decay (Tau) of [Ca]²⁺_i transients were also measured. Half width (±isoproterenol conditions) was significantly less in dnTRPC4 (sham) versus WT (sham) myocytes (dnTRPC4 [sham]±isoproterenol: 220±11 versus 209±9 ms; WT [sham]±isoproterenol: 280±20 versus 261±17 ms; 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 (±isoproterenol conditions) remained significantly less in dnTRPC4 versus WT myocytes (dnTRPC4 [MI]±isoproterenol: 247±16 versus 230±14 ms; WT [MI]±isoproterenol: 315±15 versus 279±11 ms; Figure 8C). Isoproterenol 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±isoproterenol 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 LTCC and loss of adrenergic regulation is a common feature of diseased cardiac myocytes.⁴⁷ We next examined if loss of TRPC function influenced the behavior of LTCC 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 versus WT: −13.0±1.4 versus −12.1±0.85 pA/pF; Figure 8E). Isoproterenol increased I_Ca,L density in both sham dnTRPC4 myocytes (pre-isoproterenol versus after isoproterenol: −13.0±1.4 versus −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 versus WT: −11.8±1.7 versus −10.4±1.1 pA/pF; Figure 8E). However, only dnTRPC4 myocytes showed a significant increase in I_Ca,L with isoproterenol after MI (pre-isoproterenol versus after isoproterenol: −11.8±1.7 versus −18.3±2.5 pA/pF). Cell capacitance (Figure 8F) and cell size (Online Figure XD and XE) were similar in sham WT and dnTRPC4 animals, but a significant increase in cell capacitance was seen in WT cells after MI (Figure 8F) but not in dnTRPC4 myocytes.