Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes

Abstract

Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.

Fig. 1.

Immunostaining of endogenous SERCA2a in neonatal rat (A and B) or embryonic chicken (C and D) myocytes. The myocytes were infected with adenoviral vectors containing just the U6 promoter (A and C) or the U6 promoter followed by the RNAi template (B and D). Forty-eight hours after infection, the cells were harvested for staining. Expression of endogenous SERCA2a is significantly decreased by infection with the siRNA-containing vector in both rat and chicken cardiac myocytes.

Fig. 2.

Real-time RT-PCR, Western blot, and functional analysis of endogenous SERCA2a in neonatal rat (A and C) or embryonic chicken (B and D) myocytes. RT-PCR and Western blots (A and B) as well as Ca²⁺ transport (C and D) measurements demonstrate down-regulation of endogenous SERCA2a expression in both rat and chicken cardiac myocytes after infection with adenoviral vectors containing the U6 promoter and the RNAi template. Control myocytes were infected with vectors containing the U6 promoter only. GAPDH and actin (data not shown) were used as standards for analysis of the total RNA (RT-PCR) or protein (Western blot) samples, respectively.

Fig. 3.

Myoplasmic Ca²⁺ and l-type Ca²⁺ currents in neonatal rat myocytes. (A) Average myoplasmic Ca²⁺ transients recorded during a step depolarization to 0 mV in control (Upper, n = 8) and siRNA (Lower, n = 5) myocytes. (B) Current–voltage relationship for control (○, n = 7) and siRNA (•, n = 4) myocytes. Points represent the mean ± SEM peak inward current (200 ms, P/4 leak correction) with a Na⁺-free bathing solution. (C) Ca²⁺ transients in response to a series of six voltage-clamp depolarizations (data not shown) to –10 mV at 2-s intervals. The top two records are from control myocytes in Na⁺-containing (○, n = 6) and Na⁺-free (•, n = 3) bathing solution, respectively. The two lowermost records are from siRNA myocytes in Na⁺-containing (□, n = 5) and Na⁺-free (▪, n = 4) solution. The symbols to the right of each record indicate the corresponding trace in D. Each record is normalized to the peak of its first transient. (D) The elevation of myoplasmic Ca²⁺ before each of six test pulses from records shown in C, plotted as the difference (ΔCa²⁺) from the resting level (before the first transient). Note that the Ca²⁺ does not recover completely by the time of the next pulse, with the result that diastolic Ca²⁺ slowly accumulates. The degree of accumulation is greater in siRNA myocytes (□) than in controls (○). Also, functional suppression of NCX by Na⁺-free bathing solution results in a further increase in the accumulation of diastolic Ca²⁺ of siRNA myocytes (compare □ and ▪) but not in control cells (compare ○ and •). The accumulation of diastolic Ca²⁺ reflects the compromised ability of the siRNA cells to remove myoplasmic Ca²⁺ after a transient, which is further slowed in Na⁺-free bathing solution by suppression of NCX. (E) Western blot of the expression of NCX (see Methods) in control and siRNA myocytes, showing that NCX expression is 5-fold greater in siRNA cells compared with controls.

Fig. 4.

Silencing the SERCA2a gene in neonatal rat cardiac myocytes results in increased Ba²⁺ entry through TRPCs. The myocytes were infected with empty or RNAi template vectors as described in Fig. 1. Seventy-two hours after infection, the cells, which were grown on coverslips, were loaded with fura-2 acetoxymethyl ester, and the medium was changed to one that was Ca²⁺-free and in which Na⁺ was replaced by N-methyl-d-glucamine. (A) After 8 min of exposure to 2 μM thapsigargin and 2 min of exposure to 10 μM nifedipine, 1.0 mM BaCl₂ was added by medium replacement, and the fura-2 fluorescence was monitored with an InCyt imaging system. (B) The experiments were repeated in the presence of 10 μM SKF96365. Each fluorescence trace is the average derived from 10–15 cells in each of three different experiments. The dashed lines delimit the SE for each condition. The Western blots in A demonstrate the increased expression of TRPC4 and TRPC5 protein after SERCA silencing.

Fig. 5.

Effects of silencing the SERCA2a gene in neonatal rat cardiac myocytes on various Ca²⁺ signaling genes as tested by real-time RT-PCR. (A) Infection of myocytes with an RNAi template leads to reduced SERCA transcription within 15 h after infection, with no change in the transcriptional level of other genes. (B) Seventy-two hours after infection, SERCA transcription is further reduced, whereas increased transcription of NCX (1.7-fold) and TRPCs (2- to 3-fold) is noted. Transcription levels of the Sp1 promoter (2.8-fold); in addition, MEF2 and NFATc4 transcription factors are also increased (∼2-fold). Note that the dashed lines indicate normal transcript levels for these genes. In all of the experiments, GAPDH served as an internal control for all of the PCRs carried out, and the threshold count values were normalized to GAPDH before calculating the fold change for all of the genes. The data sets for each of the genes shown were subjected to unpaired two-tailed t test. Confidence limits: NCX, 0.02; MEF2, 0.034; Sp1, 0.035; TRPC4 and TRPC5, 0.049; NFATc4, 0.055.