Simvastatin Promotes Cardiac Myocyte Relaxation in Association with Phosphorylation of Troponin I

Abstract

The number of people taking statins is set to increase across the globe due to recent changes in prescription guidelines. For example, half the US population over 40 is now eligible for these drugs, whether they have high serum cholesterol or not. With such development in policy comes a stronger need for understanding statins' myriad of effects. Surprisingly little is known about possible direct actions of statins on cardiac myocytes, although claims of a direct myocardial toxicity have been made. Here, we determine the impact of simvastatin administration (40 mg/kg/day) for 2 weeks in normocholesterolemic rats on cardiac myocyte contractile function and identify an underlying mechanism. Under basal conditions, statin treatment increased the time to half (t_0.5) relaxation without any effect on the magnitude of shortening, or the magnitude/kinetics of the [Ca²⁺]_i transient. Enhanced myocyte lusitropy could be explained by a corresponding increase in phosphorylation of troponin I (TnI) at Ser^23,24. Statin treatment increased expression of eNOS and Ser¹¹⁷⁷ phosphorylated eNOS, decreased expression of the NOS-inhibitory proteins caveolins 1 and 3, and increased (P = 0.06) NO metabolites, consistent with enhanced NO production. It is well-established that NO stimulates protein kinase G, one of the effectors of TnI phosphorylation at Ser^23,24. Trends for parallel changes in phospho-TnI, phospho-eNOS and caveolin 1 expression were seen in atrial muscle from patients taking statins. Our data are consistent with a mechanism whereby chronic statin treatment enhances TnI phosphorylation and myocyte lusitropy through increased NO bioavailability. We see no evidence of impaired function with statin treatment; the changes we document at the level of the cardiac myocyte should facilitate diastolic filling and cardiac performance.

Keywords: cardiac; caveolin; diastole; lusitropy; nitric oxide; pleiotropy; statin.

FIGURE 1

The effect of simvastatin treatment on [Ca²⁺]_i transient and shortening. (A) Representative traces and scatter plots of amplitude and kinetics of the [Ca²⁺]_i transient. Transient amplitude is expressed as % change in fura-2 ratio units (RU) over baseline. There was no difference in diastolic [Ca²⁺]_i between groups (0.967 ± 0.008 vs. 0.976 ± 0.003 RU in cells from control and statin-treated animals respectively). n = 34–35 cells from 7 to 8 animals per group. (B) Representative traces and scatter plot of amplitude and kinetics of shortening. Shortening amplitude is expressed as % of resting cell length. There was no difference in resting cell length between groups (110 ± 2 vs. 110 ± 2 μm in cells from control and statin-treated animals respectively). Plots show mean ± SEM from n = 34–48 myocytes from 7 to 8 animals per group. ^∗∗∗P < 0.001 vs. control group, Student’s t-test.

FIGURE 2

Simvastatin treatment increases phosphorylation of troponin I (TnI). Representative immunoblots (A) and scatter plots showing myocardial levels of total troponin I (B) and Ser^23/24 phosphorylated TnI (pTnI) (C) in control and statin-treated animals. Total TnI is normalized to GADPH, pTnI is normalized to total TnI. Plots show mean ± SEM from N = 6 animals per group. ^∗∗P < 0.01 vs. control group, Student’s t-test.

FIGURE 3

Simvastatin increases eNOS expression and phosphorylation. Representative immunoblots (A) and scatter plots showing myocardial levels of eNOS (B) and Ser¹¹⁷⁷ phosphorylated eNOS (p-eNOS, C) normalized to GAPDH. (D) The proportion of total eNOS phosphorylated at Ser¹¹⁷⁷ was not different between groups. (E) NO metabolites (nitrate + nitrite) measured by a fluorimetric assay. Plots show mean ± SEM from N = 6 animals per group. ^∗∗P < 0.01 vs. control group, Student’s t-test.

FIGURE 4

Simvastatin reduces expression of caveolins 1 and 3. Representative immunoblots (A) and scatter plots showing myocardial expression of Cav1 α (upper band) and β isoforms (B) and Cav3 (C). Plots show mean ± SEM from N = 6 animals per group. ^∗P < 0.05, ^∗∗P < 0.01 vs. control group, Student’s t-test.

FIGURE 5

Effect of simvastatin on Ser²³⁹-phosphorylated VASP, an index of PKG activity. (A) Representative immunoblots showing Ser²³⁹-phosphorylated VASP and VASP expression in rat ventricular myocytes ± 8-Bromo cGMP (200 μM for 15 min, as a positive control for PKG activity) and myocardial homogenates from control and statin-treated animals. (B) Ratio of p-VASP to VASP. Plot shows mean ± SEM of N = 9 animals per group.

FIGURE 6

Statin treatment for 2 weeks has no impact on serum or myocardial cholesterol levels. (A) Serum cholesterol expressed as a % change from values on day 0 (N = 12–15 animals). (B) Myocardial cholesterol, normalized to myocardial total protein (N = 10 animals). (C) Myocardial cholesterol following sucrose gradient fractionation, values are expressed as a % of total cholesterol across all fractions (N = 3 animals).

FIGURE 7

Effect of statin treatment on the response to β-adrenoceptor (AR) stimulation. (A) Percentage change from baseline in amplitude/kinetics of the [Ca²⁺]_i transient and shortening in response to β1-AR stimulation with 100 nM isoprenaline in the presence of 100 nM ICI 118,551 (n = 14–22 cells from 3 to 4 animals per group). (B) Percentage change from baseline in amplitude/kinetics of the [Ca²⁺]_i transient and shortening in response to β2-AR stimulation with 10 μM zinterol in the presence of 300 nM CGP20712A (n = 23–28 cells from 4 animals per group). Plots show mean ± SEM. ^∗P < 0.05 vs. saline-treated group, Student’s t-test.

FIGURE 8

Effect of statin treatment on TnI and Ser^23/24 phosphorylated TnI (pTnI) in human myocardium. Representative immunoblots (A) and scatter plots showing expression of total TnI normalized to GAPDH (B) and pTnI normalized to total TnI (C) in atrial muscle from statin-treated male patients (N = 14). Patients not taking statins formed the control group (N = 6). SS = standard sample to allow inter-gel comparisons (see Materials and Methods, Western blotting). Plots show mean ± SEM. ^∗P < 0.05 vs. control group (Student’s t-test).

FIGURE 9

Effect of statin treatment on proteins that regulate NO production in human myocardium. Representative immunoblots from the same gel and scatter plots showing expression of eNOS and Ser¹¹⁷⁷ phosphorylated eNOS (p-eNOS) (A,B), Cav-1 and Cav-3 (C,D) in atrial muscle from statin-treated male patients (N = 14). Male patients not taking statins formed the control group (N = 6). Plots show mean ± SEM.