Ca(2+) influx through L-type Ca(2+) channels and transient receptor potential channels activates pathological hypertrophy signaling

Abstract

Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca(2+)]. These diseases induce cardiac hypertrophy and increases in [Ca(2+)] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of "hypertrophic" [Ca(2+)] is not known and is the topic of this study. The role of Ca(2+) influx through L-type Ca(2+) channels (LTCC), T-type Ca(2+) channels (TTCC) and transient receptor potential (TRP) channels on the activation of calcineurin (Cn)-nuclear factor of activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes (AFVMs) were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca(2+) or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca(2+)-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca(2+) induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca(2+) transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca(2+) influx through LTCCs is the primary source of Ca(2+) to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca(2+) entry via LTCCs.

Figure 1

Nifedipine, SKF-96365, and Ni²⁺ have distinct effect on Ca²⁺ transients. Ca²⁺ transients were measured in NRVMs perfused with normal Tyrode s solution (Ctrl) before and after exposure to solutions containing either 10μM Nifedipine (Nif, A), 5μM SKF-96365 (B), or 50μM Ni²⁺ (C). Peak Ca²⁺ was reduced 79%, 33%, and 20% by these treatments, respectively (D). * indicates a significant treatment effect versus control. # indicates significant difference versus Nif. P<0.05. Numbers in bars represent the number of cells studied.

Figure 2

Nifedipine blocked L-type Ca²⁺ current. Ten micromolar Nif reduced L-type Ca²⁺ current by 64.03% (A, B). SKF-96365 (5μM) and Ni²⁺ (50μM) had no significant effect on peak L-type Ca²⁺ current. Nif significantly inhibited peak I_Ca-L currents (C). * indicates significant difference as compared with control. P<0.05.

Figure 3

Ca²⁺ channel inhibitors blocked Ca²⁺ influx induced NFATc3 nuclear translocation. NRVMs were infected with Ad-NFATc3-GFP and exposed to vehicle (Ctrl, A), 4mM Ca²⁺ (B), 4mM Ca²⁺ with 10μM Nifedipine (C), 4mM Ca²⁺ with 5μM SKF-96365 (D), and 4mM Ca²⁺ with 50μM Ni²⁺ (E), respectively, for 4 hours. The nuclear GFP fluorescence intensity to cytoplasmic GFP fluorescence intensity ratio was significantly higher in NRVMs exposed to 4mM Ca²⁺ than in control (F). * indicates a significant difference versus control. # indicates a significant difference versus elevated Ca²⁺ treated NRVMs. P<0.05. Scale bar equals 50 microns.

Figure 4

Calcineurin inhibitors blocked Ca²⁺-induced NFAT translocation. CsA (2μM) and FK506 (2μM) both abolished Ca²⁺-induced NFAT nuclear translocation. * indicates a significant difference versus control. # indicates a significant difference versus Ca²⁺ treated myocytes. P<0.05. Scale bar equals 50 microns.

Figure 5

TRPC3 induced NRVM hypertrophy (A, B, G). Nifedipine (10μM, C, G) and SKF-96365 (5μM, D, H) caused a significant reduction in TRPC3 induced hypertrophy while Ni²⁺ (50μM, E, G) had no effect. FK506 (2μM, F, G) inhibited TRPC3 induced hypertrophy. Cells were stained for α-actinin (red) and DAPI (blue) to identify myocytes. Cell surface areas were measured using ImageJ. * indicates a significant difference versus control. # indicates a significant difference versus Ad-TRPC3. P<0.05. Scale bar equals 50μm.

Figure 6

TRPC3 induced NFAT-GFP nuclear translocation (A, B, G). NRVMs were infected with Ad-NFATc3-GFP alone, Ad-NFATc3-GFP and Ad-TRPC3, and Ad-NFATc3-GFP and Ad-dnTRPC6 (A–G), respectively. Elevated extracellular Ca²⁺ induced NFAT translocation in control cells and increased NFAT nuclear translocation in TRPC3 cells (A, B, D, E, G). dnTRPC6 reduced Ca²⁺ induced NFAT nuclear translocation (C, F, G). * indicates a significant difference versus control. # indicates a significant difference versus Ca²⁺ treatment. P<0.05.

Figure 7

Nifedipine blocked NFAT translocation induced by TRPC3 overexpression (A, B, C, G). SKF-96365 (5μM, D) reduced TRPC3-induced NFAT nuclear translocation but was less effective than Nif. Ni²⁺ (50μM, E) had no significant effect on TRPC3 induced NFAT translocation. FK506 (2μM, F) eliminated TRPC3 induced NFAT translocation. * indicates a significant difference versus control. # indicates a significant difference versus TRPC3. P<0.05.

Figure 8

TRPC3 prolonged the Ca²⁺ transient (A). The peak of Ca²⁺ transient was not significantly altered in TRPC3-infected cells (B). The time to half decay was significantly prolonged in NRVMs infected with Ad-TRPC3 versus controls (C).