Limits...
Enhanced cardiac TBC1D10C expression lowers heart rate and enhances exercise capacity and survival

View Article: PubMed Central - PubMed

ABSTRACT

TBC1D10C is a protein previously demonstrated to bind and inhibit Ras and Calcineurin. In cardiomyocytes, also CaMKII is inhibited and all three targeted enzymes are known to promote maladaptive cardiomyocyte hypertrophy. Here, in accordance with lack of Calcineurin inhibition in vivo, we did not observe a relevant anti-hypertrophic effect despite inhibition of Ras and CaMKII. However, cardiomyocyte-specific TBC1D10C overexpressing transgenic mice exhibited enhanced longevity. Ejection fraction and exercise capacity were enhanced in transgenic mice, but shortening of isolated cardiomyocytes was not increased. This suggests longevity resulted from enhanced cardiac performance but independent of cardiomyocyte contractile force. In further search for mechanisms, a transcriptome-wide analysis revealed expressional changes in several genes pertinent to control of heart rate (HR) including Hcn4, Scn10a, Sema3a and Cacna2d2. Indeed, telemetric holter recordings demonstrated slower atrial conduction and significantly lower HR. Pharmacological reduction of HR was previously demonstrated to enhance survival in mice. Thus, in addition to inhibition of stress signaling, TBC1D10C economizes generation of cardiac output via HR reduction, enhancing exercise capacity and survival. TBC1D10C may be a new target for HR reduction and longevity.

No MeSH data available.


Examination of Ras/MAPK, CaMKII and CaN activity.(a) Ras activity was significantly reduced in TBC1D10C TG vs. WT mice (after phenylephrine (PE) stimulation) (WT, uninjected: n = 4; WT + PE: n = 14; TG + PE: n = 15; P < 0.05; Student’s t-test), whereas (b) p-MEK1/2/t-MEK1/2 levels were not significantly diminished in PE stimulated TG vs. PE stimulated WT mice (WT, uninjected: n = 3; WT + PE: n = 15; TG, uninjected: n = 3; TG + PE: n = 15,n.s.; Student’s t-test). (c) Calcineurin (CaN) phosphatase activity was not different between WT and TG mice (n = 6 per group), suggesting CaN-independent signal pathways may be more important for the effects of TBC1D10C in vivo. (d) Mcip-1 (Modulatory calcineurin-interacting protein 1) mRNA expression, a marker for CaN activity, was not significantly changed in TG mice whereas (e) CaMKII (T286) phosphorylation was significantly diminished in TG mice (TG: n = 8; WT: n = 10; P < 0.01; Student’s t-test). Full length blots of the cropped membranes in Fig. 4b and e are displayed in Supplementary Figure 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036039&req=5

f4: Examination of Ras/MAPK, CaMKII and CaN activity.(a) Ras activity was significantly reduced in TBC1D10C TG vs. WT mice (after phenylephrine (PE) stimulation) (WT, uninjected: n = 4; WT + PE: n = 14; TG + PE: n = 15; P < 0.05; Student’s t-test), whereas (b) p-MEK1/2/t-MEK1/2 levels were not significantly diminished in PE stimulated TG vs. PE stimulated WT mice (WT, uninjected: n = 3; WT + PE: n = 15; TG, uninjected: n = 3; TG + PE: n = 15,n.s.; Student’s t-test). (c) Calcineurin (CaN) phosphatase activity was not different between WT and TG mice (n = 6 per group), suggesting CaN-independent signal pathways may be more important for the effects of TBC1D10C in vivo. (d) Mcip-1 (Modulatory calcineurin-interacting protein 1) mRNA expression, a marker for CaN activity, was not significantly changed in TG mice whereas (e) CaMKII (T286) phosphorylation was significantly diminished in TG mice (TG: n = 8; WT: n = 10; P < 0.01; Student’s t-test). Full length blots of the cropped membranes in Fig. 4b and e are displayed in Supplementary Figure 6.

Mentions: In view of our in vitro data and previously published work demonstrating that TBC1D10C overexpression inhibits Ras/MAPK, CaMKII, and CaN, we addressed the lack of an physiologically significant antihypertrophic effect in our models by validating these signalling pathways also in vivo. We observed decreased Ras activity in TBC1D10C TG mice compared to WT littermates after 15 min of stimulation with phenylephrine (GTP-Ras/total Ras: WT uninjected: 0.78 ± 0.08, n = 4; WT+PE: 1.08 ± 0.09, n = 14; TG+PE: 0.79 ± 0.08, n = 15; P < 0.05). However, diminished Ras activity did not provoke a significant change in MEK1/2 phosphorylation (Fig. 4a,b). Furthermore, TBC1D10C TG mice did not exhibit decreased CaN activity (Fig. 4c) or altered Mcip-1 mRNA expression (no intervention, with TAC or with myocardial infarction) as a marker for CaN activity (Fig. 4d). Instead, CaMKII (T286) phosphorylation was significantly reduced in TBC1D10C TG mice (WT: 1.2 ± 0.1, n = 8; TG: 0.81 ± 0.09, n = 10; P < 0.01) (Fig. 4e). Thus, in our TG mice, we did not identify significant inhibition of either CaN or MEK1/2, suggesting this explains the lack of protection against hypertrophy. Notably, inhibition of both Ras and CaMKII activities appear as robust findings on enhanced TBC1D10C expression regardless of the method of overexpression.


Enhanced cardiac TBC1D10C expression lowers heart rate and enhances exercise capacity and survival
Examination of Ras/MAPK, CaMKII and CaN activity.(a) Ras activity was significantly reduced in TBC1D10C TG vs. WT mice (after phenylephrine (PE) stimulation) (WT, uninjected: n = 4; WT + PE: n = 14; TG + PE: n = 15; P < 0.05; Student’s t-test), whereas (b) p-MEK1/2/t-MEK1/2 levels were not significantly diminished in PE stimulated TG vs. PE stimulated WT mice (WT, uninjected: n = 3; WT + PE: n = 15; TG, uninjected: n = 3; TG + PE: n = 15,n.s.; Student’s t-test). (c) Calcineurin (CaN) phosphatase activity was not different between WT and TG mice (n = 6 per group), suggesting CaN-independent signal pathways may be more important for the effects of TBC1D10C in vivo. (d) Mcip-1 (Modulatory calcineurin-interacting protein 1) mRNA expression, a marker for CaN activity, was not significantly changed in TG mice whereas (e) CaMKII (T286) phosphorylation was significantly diminished in TG mice (TG: n = 8; WT: n = 10; P < 0.01; Student’s t-test). Full length blots of the cropped membranes in Fig. 4b and e are displayed in Supplementary Figure 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036039&req=5

f4: Examination of Ras/MAPK, CaMKII and CaN activity.(a) Ras activity was significantly reduced in TBC1D10C TG vs. WT mice (after phenylephrine (PE) stimulation) (WT, uninjected: n = 4; WT + PE: n = 14; TG + PE: n = 15; P < 0.05; Student’s t-test), whereas (b) p-MEK1/2/t-MEK1/2 levels were not significantly diminished in PE stimulated TG vs. PE stimulated WT mice (WT, uninjected: n = 3; WT + PE: n = 15; TG, uninjected: n = 3; TG + PE: n = 15,n.s.; Student’s t-test). (c) Calcineurin (CaN) phosphatase activity was not different between WT and TG mice (n = 6 per group), suggesting CaN-independent signal pathways may be more important for the effects of TBC1D10C in vivo. (d) Mcip-1 (Modulatory calcineurin-interacting protein 1) mRNA expression, a marker for CaN activity, was not significantly changed in TG mice whereas (e) CaMKII (T286) phosphorylation was significantly diminished in TG mice (TG: n = 8; WT: n = 10; P < 0.01; Student’s t-test). Full length blots of the cropped membranes in Fig. 4b and e are displayed in Supplementary Figure 6.
Mentions: In view of our in vitro data and previously published work demonstrating that TBC1D10C overexpression inhibits Ras/MAPK, CaMKII, and CaN, we addressed the lack of an physiologically significant antihypertrophic effect in our models by validating these signalling pathways also in vivo. We observed decreased Ras activity in TBC1D10C TG mice compared to WT littermates after 15 min of stimulation with phenylephrine (GTP-Ras/total Ras: WT uninjected: 0.78 ± 0.08, n = 4; WT+PE: 1.08 ± 0.09, n = 14; TG+PE: 0.79 ± 0.08, n = 15; P < 0.05). However, diminished Ras activity did not provoke a significant change in MEK1/2 phosphorylation (Fig. 4a,b). Furthermore, TBC1D10C TG mice did not exhibit decreased CaN activity (Fig. 4c) or altered Mcip-1 mRNA expression (no intervention, with TAC or with myocardial infarction) as a marker for CaN activity (Fig. 4d). Instead, CaMKII (T286) phosphorylation was significantly reduced in TBC1D10C TG mice (WT: 1.2 ± 0.1, n = 8; TG: 0.81 ± 0.09, n = 10; P < 0.01) (Fig. 4e). Thus, in our TG mice, we did not identify significant inhibition of either CaN or MEK1/2, suggesting this explains the lack of protection against hypertrophy. Notably, inhibition of both Ras and CaMKII activities appear as robust findings on enhanced TBC1D10C expression regardless of the method of overexpression.

View Article: PubMed Central - PubMed

ABSTRACT

TBC1D10C is a protein previously demonstrated to bind and inhibit Ras and Calcineurin. In cardiomyocytes, also CaMKII is inhibited and all three targeted enzymes are known to promote maladaptive cardiomyocyte hypertrophy. Here, in accordance with lack of Calcineurin inhibition in vivo, we did not observe a relevant anti-hypertrophic effect despite inhibition of Ras and CaMKII. However, cardiomyocyte-specific TBC1D10C overexpressing transgenic mice exhibited enhanced longevity. Ejection fraction and exercise capacity were enhanced in transgenic mice, but shortening of isolated cardiomyocytes was not increased. This suggests longevity resulted from enhanced cardiac performance but independent of cardiomyocyte contractile force. In further search for mechanisms, a transcriptome-wide analysis revealed expressional changes in several genes pertinent to control of heart rate (HR) including Hcn4, Scn10a, Sema3a and Cacna2d2. Indeed, telemetric holter recordings demonstrated slower atrial conduction and significantly lower HR. Pharmacological reduction of HR was previously demonstrated to enhance survival in mice. Thus, in addition to inhibition of stress signaling, TBC1D10C economizes generation of cardiac output via HR reduction, enhancing exercise capacity and survival. TBC1D10C may be a new target for HR reduction and longevity.

No MeSH data available.