Ca2+/calmodulin-dependent protein kinase II and protein kinase G oxidation contributes to impaired sarcomeric proteins in hypertrophy model

Abstract

Aims: Volume overload (VO) induced hypertrophy is one of the hallmarks to the development of heart diseases. Understanding the compensatory mechanisms involved in this process might help preventing the disease progression.

Methods and results: Therefore, the present study used 2 months old Wistar rats, which underwent an aortocaval fistula to develop VO-induced hypertrophy. The animals were subdivided into four different groups, two sham operated animals served as age-matched controls and two groups with aortocaval fistula. Echocardiography was performed prior termination after 4- and 8-month. Functional and molecular changes of several sarcomeric proteins and their signalling pathways involved in the regulation and modulation of cardiomyocyte function were investigated.

Results: The model was characterized with preserved ejection fraction in all groups and with elevated heart/body weight ratio, left/right ventricular and atrial weight at 4- and 8-month, which indicates VO-induced hypertrophy. In addition, 8-months groups showed increased left ventricular internal diameter during diastole, RV internal diameter, stroke volume and velocity-time index compared with their age-matched controls. These changes were accompanied by increased Ca²⁺ sensitivity and titin-based cardiomyocyte stiffness in 8-month VO rats compared with other groups. The altered cardiomyocyte mechanics was associated with phosphorylation deficit of sarcomeric proteins cardiac troponin I, myosin binding protein C and titin, also accompanied with impaired signalling pathways involved in phosphorylation of these sarcomeric proteins in 8-month VO rats compared with age-matched control group. Impaired protein phosphorylation status and dysregulated signalling pathways were associated with significant alterations in the oxidative status of both kinases CaMKII and PKG explaining by this the elevated Ca²⁺ sensitivity and titin-based cardiomyocyte stiffness and perhaps the development of hypertrophy.

Conclusions: Our findings showed VO-induced cardiomyocyte dysfunction via deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative state of CaMKII and PKG and this might contribute to the development of hypertrophy.

Keywords: Hypertrophy; Oxidative stress; Sarcomeric proteins; Volume-overload.

Figure 1

Echocardiographic data. (A) Experimental set up, (B) ejection fraction, (C) LVID: left ventricule internal diameter in/during diastole, (D) RVID: right ventricle internal diameter, (E) stroke volume, (F) velocity‐time index, (G) MABP: mean arterial blood pressure. Data are shown as mean ± SEM; n = 5–6. *P < 0.05. 4‐month versus 8‐month and #P < 0.05 Ctrl versus VO using One way ANOVA Tukey multiple comparisons test.

Figure 2

Organ weights. (A) Animals' weights, (B) heart weights, (C) heart/body weight ratio, (D) left ventricular weights, (E) right ventricular weights, and (F) atria weights. Data are shown as mean ± SEM; n = 5–6. *P < 0.05. 4‐month versus 8‐month and #P < 0.05 Ctrl versus VO using one‐way ANOVA Tukey multiple comparisons test.

Figure 3

Sarcomeric protein phosphorylation. (A) Calcium sensitivity, (B) maximum tension of sarcomere. Data are shown as mean ± SEM; n = 5–6. n = 5–6. *P < 0.05. Ctrl 4‐month versus 8‐month; †P < 0.05/†††P < 0.001 Ctrl versus VO 8‐month; ‡‡‡P < 0.001 4‐month versus 8‐month VO; and #P < 0.05 Ctrl versus VO 4‐month using one‐way ANOVA Tukey multiple comparisons test. (C) Phosphorylation of cardiac myosin binding protein C level. (D) Site‐specific phosphorylation of cTnI Ser_23/24. (E) Site‐specific phosphorylation of cTnI Ser₄₃. (F) Site‐specific phosphorylation of cTnI Thr₁₄₃. Data are shown as mean ± SEM; n = 5–6. *P < 0.05 4‐month versus 8‐month, #P < 0.05 Ctrl versus VO using One way ANOVA Tukey multiple comparisons test.

Figure 4

Titin‐based myocardial stiffness and titin phosphorylation by CaMKII. (A) Passive tension‐sarcomere length relations of control (Ctrl) and volume overload (VO) cardiomyocytes at sarcomere length (SL) 1.8–2.4 μM. All force recordings were normalized for cardiomyocyte cross‐sectional area. Curves represent third order polynomial regressions. **P < 0.01/***P < 0.001. Ctrl 4‐month versus 8‐month; †P < 0.05/†††P < 0.001 Ctrl versus VO 8‐month; ‡‡P < 0.01/‡‡‡P < 0.001 4‐month versus 8‐month VO; and #P < 0.05/##P < 0.01 Ctrl versus VO 4‐month using one‐way ANOVA Tukey multiple comparisons test. (B) Overall titin and (E–G) CaMKII‐specific phosphorylation measurement by western immunoblots from left ventricular tissue. Representative blots show antibody staining at position (C) Ser₄₀₄₃ (N2Bus) and (D) Ser₁₂₈₈₄ (PEVK). PVDF stains are included for comparisons of loading (E) oxidation of CaMKII, (F) CaMKII expression level, (G) phosphorylation of CaMKII at Thr₂₈₆. Data are shown as mean ± SEM; n = 4. #P < 0.05 Ctrl versus VO using one‐way ANOVA Tukey multiple comparisons test.

Figure 5

Titin phosphorylation by PKG. (A) Titin phosphorylation at Ser₄₀₈₀ (N2Bus), (B) representative blot of PKG levels. (C) Distribution of PKG isoforms, (D) monomer level of PKG, (E) dimer level of PKG, (F) polymer level of PKG. Data are shown as mean ± SEM; n = 5–6. #P < 0.05 Ctrl versus VO. *P < 0.05 4‐month versus 8‐month using one‐way ANOVA Tukey multiple comparisons test.