RBFOX1 Regulates Calcium Signaling and Enhances SERCA2 Translation

Abstract

RBFOX1 is an RNA-binding protein that regulates alternative splicing and RNA processing in the neurons, skeletal muscle, and heart. We intended to define the role of RBFOX1 in regulating calcium homeostasis to maintain normal cardiac function. We generated cardiomyocyte-specific Rbfox1 gene-deletion mice (cKO). The cardiomyocyte-specific deletion of RBFOX1 was confirmed by Western blotting and immunohistochemistry. The cKO mice showed mild hypertrophy and depressed cardiac function under homeostatic conditions, which did not deteriorate with age. Pressure overload by trans-aortic constriction (TAC) caused exaggerated cardiac hypertrophy and accelerated heart failure in cKO compared with wild-type mice. We performed Western blotting to assess the expression of important Ca²⁺-handling proteins, which showed alterations in the phosphorylation of PLN and CAMKII and decreased expression of SERCA2. We measured the Ca²⁺ dynamics and noted significantly delayed Ca²⁺ reuptake into the sarcoplasmic reticulum. Importantly, the decrease in SERCA2 expression was not due to reduced mRNA expression or altered splicing. To assess the possibility of the post-transcriptional regulation of SERCA2 expression by RBFOX1, we performed RNA immunoprecipitation (RIP), which showed the binding of RBFOX1 protein to Serca2 mRNA, which was confirmed in luciferase assays with the Serca2a 3'-untranslated region fused to luciferase. Finally, we performed a puromycin incorporation experiment, which showed that RBFOX1 enhances SERCA2 protein translation. Our results show that RBFOX1 plays a crucial role in regulating the expression of Ca²⁺-handling genes to maintain normal cardiac function. We show an important post-transcriptional role of RBFOX1 in regulating SERCA2 expression.

Keywords: calcium signaling; cardiac hypertrophy; gene regulation; heart failure.

Figure 1

Cardiac specific deletion of Rbfox1 alters calcium handling protein expression. (A). Immunostaining of cardiac sections with RBFOX1 (Red) DAPI (blue) and Desmin (Green) from control (CON) and cardiomyocyte-specific Rbfox1 knock-out mice (cKO), bar = 25 µm (B). Echocardiographic assessment of Left Ventricular Ejection Fraction (LVEF) in Control and cKO mice (n = 5 and 6). (C). Analysis of heart weight/body weight ratio (HW/BW) in Control and cKO mice (n = 5 and 6). (D). Representative staining of wheat-germ agglutinin to measure histological cross-sectional area of cardiomyocytes with quantification, bar = 100 µm (n = 3 and 3) (E). Western blot analysis of calcium handling proteins; pospho-PLN (ser16 and thr17), PLN, SERCA2, NCX, oxidized-CAMKII, phospho-CAMKII (thr286), CAMKII, phospho-Troponin I (ser23/24), and Troponin I in heart lysates of 1-month old mice. GAPDH was used as a loading control. (F). Representative immunostaining for SERCA2 (red) and DAPI (blue) with quantification of SERCA2 intensity, bar = 100 µm (n = 3 and 3).

Figure 2

Cardiac specific deletion of Rbfox1 alters calcium handling protein expression and causes mild cardiac dysfunction. (A). Echocardiographic assessment of Left Ventricular Ejection Fraction (LVEF) in Control and cKO mice (n = 9 and 11). (B). Analysis of heart weight/body weight ratio (HW/BW) in Control and cKO mice (n = 6 and 7). (C). Western blot analysis of calcium handling proteins; p-PLN (ser16 and thr17), PLN, SERCA2, NCX, oxi-CAMKII, p-CAMKII (thr286), CAMKII, p-Troponin I (ser23/24), and Troponin I in heart lysates of 1-month old mice. GAPDH was used as a loading control. (D). Electron microscopy of control and cKO mouse hearts. Bar = 1 µm. * p < 0.05 vs. Control.

Figure 3

Rbfox1 cKO mice display mild cardiac dysfunction and apoptosis with aging. (A). Echocardiographic assessment of left ventricular ejection fraction (LVEF) in Control and cKO mice at 12 month of age (n = 10 and 6). (B). Analysis of heart weight/body weight ratio in Control and cKO mice at 12 months of age (n = 10 and 6). (C). Analysis of minimum fiber diameter (MFD) of cardiomyocytes from histological sections (n = 6 and 6). (D). Quantification of cleaved caspase-3 staining within cardiomyocytes, indicative of cardiomyocyte apoptosis (n = 6 and 6). (E). Above: Representative images of fast green, Sirius red staining for collagen deposition in Control and cKO mice (bar = 100 µm). Below: Quantification of collagen in control and cKO mice (n = 6 and 6) * p < 0.05 vs. Control.

Figure 4

Rbfox1 cKO mice are sensitive to TAC-induced cardiac dysfunction and show dysregulation of calcium handling proteins. (A). Echocardiographic assessment of left ventricular ejection fraction (LVEF) in Control and cKO mice 2 weeks after sham or TAC surgery (n = 5, 9, 6, 9). (B). Analysis of heart weight/body weight ratio in Control and cKO mice 2 weeks after sham or TAC surgery (n = 5, 8, 7, 9). (C). Minimal fiber diameter of cardiomyocytes from histological sections of control and cKO mice after sham or TAC surgery (n = 3, 6, 7, 9). (D). Quantification of cardiomyocyte cleaved caspase-3 staining in Control and cKO mice 2 weeks after sham or TAC surgery. Image shows example of cardiomyocyte that is positive for cleaved caspase-3 staining (white arrow, bar = 10 µm). (E). Fast green, Sirius red staining for collagen deposition in Control and cKO mice 2 weeks after sham or TAC surgery. Images show representative examples (bar = 100 µm). (F). Western blot analysis of Rbfox1 in Control and cKO mice 2 weeks after sham or TAC surgery. (G). Western blot analysis of calcium handling proteins; p-PLN (ser16 and thr17), PLN, SERCA2, NCX, oxi-CAMKII, p-CAMKII (thr286), CAMKII, p-Troponin I (ser23/24), and Troponin I in heart lysates of Control and cKO mice 2 weeks after sham or TAC surgery. GAPDH was used as a loading control. * p < 0.05 vs. sham † p < 0.05 vs. Control TAC.

Figure 5

RBOFX1 regulates calcium dynamics. (A). Resting Ca²⁺ level measured by Fura2 ratio (340/380) in cardiomyocytes isolated from Control and cKO mice (n = 3 and 4 mice). (B). Rise in Ca²⁺ level upon paced transient measured by Fura2 ratio (340/380) in cardiomyocytes isolated from Control and cKO mice (n = 3 and 4 mice). (C). Decline Ca²⁺ level measured by Fura2 ratio (340/380) in cardiomyocytes isolated from Control and cKO mice (n = 3 and 4 mice). (D). Design of PCR primers to detect overall Serca2 mRNA levels (exon 1-3) or Serca2a vs. Serca2b specific isoforms. (E). qPCR for total Serca2 mRNA expression from Control and cKO hearts at 3 months of age (n = 5 and 5). (F). qPCR for total Serca2a mRNA expression from Control and cKO hearts at 3 months of age (n = 5 and 5). (G). qPCR for total Serca2b mRNA expression from Control and cKO hearts at 3 months of age (n = 5 and 5). * p < 0.05 vs. Control.

Figure 6

RBFOX1 regulates Serca2 mRNA translation. (A). Western blot analysis of RBFOX1, SERCA2 and GAPDH protein in neonatal rat ventricular cardiomyocytes after transfection with Rbfox1 or control siRNA. (B). Densitometry analysis of Rbfox1 expression corrected to GAPDH after control or Rbfox1 siRNA transfection (n = 3 and 3). (C). Densitometry analysis of SERCA2 expression after control or Rbfox1 siRNA transfection (n = 3 and 3). (D). qPCR expression of Serca2 mRNA after RNA immunoprecipitation using RBFOX1 antibody in heart lysates of Control and Rbfox1 cKO mice (n = 5 and 5). (E). Western blot for incorporated puromycin in proteins extracted from neonatal rat ventricular cardiomyocytes after transfection with Rbfox1 or control siRNA. (F). Western blot for SERCA2 from protein lysates after immunoprecipitation using puromycin antibody. (G). Densitometry analysis of newly formed SERCA2 protein based on puromycin immunoprecipitation in panel (F). (n = 5 and 5). (H). Dual luciferase assay for Luciferase cDNA plasmid with Serca2a 3′UTR or mutated Serca2 3′UTR, where RBFOX1 binding sites have been mutated, with or without Rbfox1 expression vector co-transfection (n = 6, 12 and 12). * p < 0.05 vs. Control † p < 0.05 vs. Serca2a 3′UTR with Rbfox1.