Titin based viscosity in ventricular physiology: an integrative investigation of PEVK-actin interactions

Abstract

Viscosity is proposed to modulate diastolic function, but only limited understanding of the source(s) of viscosity exists. In vitro experiments have shown that the proline-glutamic acid-valine-lysine (PEVK) rich element of titin interacts with actin, causing a viscous force in the sarcomere. It is unknown whether this mechanism contributes to viscosity in vivo. We tested the hypothesis that PEVK-actin interaction causes cardiac viscosity and is important in vivo via an integrative physiological study on a unique PEVK knockout (KO) model. Both skinned cardiomyocytes and papillary muscle fibers were isolated from wildtype (WT) and PEVK KO mice and passive viscosity was examined using stretch-hold-release and sinusoidal analysis. Viscosity was reduced by ~60% in KO myocytes and ~50% in muscle fibers at room temperature. The PEVK-actin interaction was not modulated by temperature or diastolic calcium, but was increased by lattice compression. Stretch-hold and sinusoidal frequency protocols on intact isolated mouse hearts showed a smaller, 30-40% reduction in viscosity, possibly due to actomyosin interactions, and showed that microtubules did not contribute to viscosity. Transmitral Doppler echocardiography similarly revealed a 40% decrease in LV chamber viscosity in the PEVK KO in vivo. This integrative study is the first to quantify the influence of a specific molecular (PEVK-actin) viscosity in vivo and shows that PEVK-actin interactions are an important physiological source of viscosity.

Figure 1

Viscosity in skinned cardiomyocytes. A) Example of stretch-hold-release experiments for WT (black) and KO (gray) cardiomyocytes stretched at different speeds. Stress relaxation during the hold phase is less pronounced in the KO cells (arrows). B) KO (gray) cells have a significantly reduced viscous stress at all speeds and a 67% reduction in the coefficient of viscosity (slope) compared to WT (black). Inset: Schematic of how viscous stress (σ_v) is defined. C) KO (gray) cells have smaller viscous moduli than WT (black) cells. (Every third error bar (SE) plotted for clarity.) Inset: Schematic showing how the viscous modulus (VM) is calculated from the magnitude of the stress (σ, gray), the length change (ε, black) and the phase delay (θ) between steady state stress and length traces. (Length change was normalized to prep length and viscous modulus is in stress per fractional length change).

Figure 2

Viscosity in skinned cardiac tissue. Skinned WT and KO papillary muscle fibers were stretched at 4 speeds. In (A) the slowest (0.1 length/s; black) and fastest (50 lengths/s; gray) speeds are shown superimposed for WT (left) and KO (right) tissues with WT showing a higher speed dependence of viscous stress-relaxation (Arrows). B) KO tissues (gray) have a reduced viscous stress and a 47% reduction in the coefficient of viscosity compared to WT (black) tissues. Myofilament extraction reveals almost no viscous phenotype for the ECM. C) Viscous moduli are significantly reduced in the KO vs. WT tissues, but no ECM effect. (Viscous modulus was normalized to slack fiber length).

Figure 3

A) Effect of actomyosin inhibitor blebbistatin on passive viscosity of skinned muscle at room (RT, 24°C) and physiological temperature (37°C). At RT blebbistatin has no effect on viscosity. Physiological temperature increases viscosity but this increase is abolished by blebbistatin. Finding are the same in WT (left) and PEVK KO (right) tissues. * p<0.05 vs RT -.

Figure 4

Diastolic viscosity in isolated hearts. A) Typical stretch-hold-release experiment in heart; viscous stress was calculated as the peak minus stress at end of hold. B) KO (gray) hearts have a 32% reduction in viscous stress compare to WT (black,*p=0.01). C) Example sinusoidal oscillation (10Hz) induced on the beating heart; viscous modulus was calculated from the oscillation during the diastolic plateau (box). D) Viscous moduli calculated at 6 frequencies during a diastolic plateau. KO hearts show an average 43% reduction in viscous moduli compared to WT.

Figure 5

Viscosity in-vivo. Transmitral Doppler from WT (A) and KO (B) hearts show an asymmetric E-wave for the WT and a more symmetric E-wave in the KO. AT (gray line) is shorter in WT (12ms) than KO (19ms) while DT (black line) is longer in WT (29ms) than KO (25ms). Characterizing the same WT (C) and KO (D) hearts using a viscoelastic model indicates that the WT has a prolonged deceleration phenotype (tail) while the KO has a more symmetric shape that corresponds to a reduced viscosity. (Vertical scale lines denote 50cm/s velocity. Images contrast-adjusted for clarity.)