The CaMKII phosphorylation site Thr1604 in the CaV1.2 channel is involved in pathological myocardial hypertrophy in rats

Figures

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  Figure 1.. Expression of members of the CaMKII/HDAC/MEF2/ANP pathway and the effect of AIP on ISO-induced cardiac hypertrophy in a cardiomyocyte model. (a) Representative CaMKII expression and quantification. (b) p-CaMKII expression in each group. (c) HDAC expression in each group. (d) The expression of p-HDAC in each group. (e) MEF2 C expression in the cardiomyocyte hypertrophy cell model. (f) Representative ANP expression and quantification. n = 6, *P < 0.05, **P < 0.01, and ***P < 0.001, ISO group vs control group; #P < 0.05 and ###P < 0.001, AIP group vs ISO group. AIP: autocamtide-2-related inhibitory peptide, HDAC: histone deacetylase. MEF: myocyte-specific enhancer factor.

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  Figure 2.. CaV1.2 protein expression in an ISO-induced myocardial hypertrophy model and CaMKII-induced phosphorylation of CaV1.2 (Thr1604). (a) The location of the Thr1604 CaMKII phosphorylation site on the CaV1.2 channel C-terminus (GenBank: AC_000072.1, NP_036649.2 rat heart). (b) and (c) Immunoblot assay of CaV1.2 and p-CaV1.2 (Thr1604) protein levels in homogenates obtained from ISO-induced cultured neonatal rat cardiomyocytes. (d) Coimmunoprecipitation of p-CaV1.2 (Thr1604) and CaMKII in cardiomyocytes (The bands containing CaMKII and p-CaV1.2 were normalized to that of precipitated CaV1.2 to measure CaMKII-induced CaV1.2 phosphorylation at Thr1604.) n = 6, *P < 0.05 and **P < 0.01 vs control group, #P < 0.05 and ###P < 0.001 AIP group vs ISO group. AIP: autocamtide-2-related inhibitory peptide.

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  Figure 3.. Persistent ISO stimulation induced cardiac hypertrophy in SD rats. (a) Heart weight and left ventricular weight normalized to body weight. BW: body weight, HW: heart weight, LVW: left ventricular weight. **P < 0.01 and ***P < 0.001 compared with the control group. ##P < 0.01 and ###P < 0.001 compared with the ISO 2-week group. Each group contained 6–7 rats. (b) Hematoxylin- and eosin-stained sections and cardiomyocyte cross-sectional areas in each group. Average of 10 stained sections for each heart, n = 3 hearts in each group. *P < 0.05 and ***P < 0.001 vs the control group; ###P < 0.001, ISO 2-week group vs ISO 3-week group.

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  Figure 4.. CaMKII/HDAC/MEF2/ANP expression in myocardial hypertrophy development in an SD rat model. (a) Representative CaMKII expression and quantification. (b) p-CaMKII expression in each group. (c) HDAC expression in each group. (d) The expression of p-HDAC in each group. (e) MEF2 C expression in the cardiac hypertrophy model. (f) Representative ANP expression and quantification; n = 6, *P < 0.05, **P < 0.01, and ***P < 0.001, ISO group vs control group; #P < 0.05, ISO 2-week group vs ISO 3-week group.

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  Figure 5.. CaV1.2 protein expression and CaMKII-mediated phosphorylation of CaV1.2 at the Thr1604 site in the development of myocardial hypertrophy. (a) and (b) Immunoblot assay to detect the CaV1.2 and p-CaV1.2 (Thr1604) proteins in left ventricular homogenates obtained from ISO-induced cardiac hypertrophy rats. (C, D, E) Coimmunoprecipitation of p-CaV1.2 (Thr1604) and CaMKII in homogenates. (The bands containing CaMKII and p-CaV1.2 in each group were normalized to that of precipitated CaV1.2 to measure the CaMKII-induced CaV1.2 phosphorylation at Thr1604.) n = 6, *P < 0.05, **P < 0.01, and ***P < 0.001 vs control group, #P < 0.05 ISO 2-week group vs ISO 3-week group.

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  Figure 6.. A schematic of the hypothesis that CaMKII-induced CaV1.2 channel phosphorylation at the Thr1604 site induces pathological myocardial hypertrophy development in rats. Briefly, a signaling cascade after ISO-dependent stimulation of cardiomyocyte β-ARs involves the activation of Gs proteins, which in turn stimulate cyclic adenosine monophosphate (cAMP). Exchange protein directly activated by cAMP (Epac) activates CaMKII-mediated phosphorylation of CaV1.2 at the C-terminal Thr1604 site, opening the CaV1.2 channel and increasing intracellular calcium levels. The latter further promotes CaMKII autophosphorylation. This positive feedback mechanism enhances the CaMKII/HDAC/ANP hypertrophic signaling pathway. Moreover, elevated intracellular calcium may also act on the β-AR/PKA hypertrophic signaling pathway, as reported by the group of William A. Catterall [29,30,33], eventually leading to irreversible cardiac hypertrophy and heart failure. β-AR: β-adrenergic receptor; cAMP: cyclic adenosine monophosphate; Epac: exchange protein directly activated by cAMP; CaMKII: Ca2+/calmodulin-dependent protein kinase II; HDAC: histone deacetylase; PKA: protein kinase A; CaN: calcineurin; NFAT:…