Calpain inhibition preserves talin and attenuates right heart failure in acute pulmonary hypertension

Abstract

Right heart failure from right ventricular (RV) pressure overload is a major cause of morbidity and mortality, but its mechanism is incompletely understood. We tested the hypothesis that right heart failure during 4 hours of RV pressure overload is associated with alterations of the focal adhesion protein talin, and that the inhibition of calpain attenuates RV dysfunction and preserves RV talin. Anesthetized open-chest pigs treated with the calpain inhibitor MDL-28170 (n = 20) or inactive vehicle (n = 23) underwent 4 hours of RV pressure overload by pulmonary artery constriction (initial RV systolic pressure, 64 ± 1 and 66 ± 1 mm Hg in MDL-28170 and vehicle-treated pigs, respectively). Progressive RV contractile dysfunction was attenuated by MDL-28170: after 4 hours of RV pressure overload, RV systolic pressure was 44 ± 4 mm Hg versus 49 ± 6 mm Hg (P = 0.011), and RV stroke work was 72 ± 5% of baseline versus 90 ± 5% of baseline, (P = 0.027), in vehicle-treated versus MDL-28170-treated pigs, respectively. MDL-28170 reduced the incidence of hemodynamic instability (death or systolic blood pressure of < 85 mm Hg) by 46% (P = 0.013). RV pressure overload disrupted talin organization. MDL-28170 preserved talin abundance in the RV free wall (P = 0.039), and talin abundance correlated with the maintenance of RV free wall stroke work (r = 0.58, P = 0.0039). α-actinin and vinculin showed similar changes according to immunohistology. Right heart failure from acute RV pressure overload is associated with reduced talin abundance and disrupted talin organization. Calpain inhibition preserves the abundance and organization of talin and RV function. Calpain inhibition may offer clinical utility in treating acute cor pulmonale.

Figure 1.

Instrumentation of the heart. A solid-state micromanometer catheter was introduced into the right ventricle (RV) via an internal jugular vein. An array of four sonomicrometry crystals (indicated by x) was inserted into the mid-RV free wall and used to calculate wall area (see online supplement). Chord lengths used to calculate area are designated by a, b, c, d, p, and q. Ultrasonic transit-time flow probes were placed around the main pulmonary artery for the measurement of cardiac output, and around the proximal right coronary artery for the measurement of coronary artery flow. An umbilical tape snare was placed around the pulmonary artery to produce right ventricular pressure overload (RVPO), and a hydraulic occluder was placed around the inferior vena cava to alter preload. In addition to the instrumentation illustrated here, a micromanometer catheter was inserted into the left ventricle via apical puncture, pacing wires were affixed to the left atrial appendage, and a 26-gauge catheter was inserted into the proximal right coronary artery. A lead II electrocardiogram was recorded from needle electrodes placed subcutaneously in the four limbs. Ao, aorta; RA, right atrium; PA, pulmonary artery; RV, right ventricle; IVC, inferior vena cava.

Figure 2.

Experimental protocol. Vertical arrows indicate the approximate time at which hemodynamic measurements were obtained. Treatment assignment was blinded during the experimental protocol by randomly selecting coded vials of solutions of vehicle (VEH) or MDL-28170 (MDL) that had been prepared in advance by an investigator not directly involved in the animal experiments. After baseline measurements of hemodynamics and RV function, an infusion of either inactive vehicle or MDL-28170 was begun and a second set of measurements of hemodynamics and RV function was repeated 20 minutes later to determine any effects of treatment in the absence of RVPO (designated Infusion). Next, the pulmonary artery umbilical tape snare was gradually constricted for 5–10 minutes to obtain maximum RV systolic pressure (the point at which additional pulmonary artery constriction resulted in a decline in RV systolic pressure). This degree of pulmonary artery constriction was fixed for the ensuing 4 hours to simulate clinical conditions of increased pulmonary vascular resistance. Additional sets of hemodynamic data were obtained early (10–20 min, designated RVPO 20 min) and later (230–240 min, designated RVPO 4 hrs) after beginning pulmonary artery constriction. In the 35 pigs surviving until the end of the 4 hours of RVPO, the snare was released and “recovery” measurements were obtained 10–20 minutes later. In approximately half of the pigs, the heart was arrested with 10% KCl, and the central RV free wall was excised and frozen on a liquid nitrogen–cooled steel mortar for later analysis. Tissue from pigs that died before the end of the protocol was not analyzed, because low perfusion pressure in those pigs could have caused antemortem ischemia and resulted in nonspecific protein degradation.

Figure 3.

Examples of hemodynamic data obtained in a pig treated with inert vehicle that developed RV pulsus alternans near the end of 4 hours of RVPO. Prominent alterations of RV pressure, RV free wall contraction, and pulmonary artery (PA) flow occurred. The minimal concurrent alternation of left ventricular (LV) pressure was likely attributable to interventricular interactions. The electrocardiogram (ECG) indicates atrial paced rhythm. Treatment with MDL-28170, compared with vehicle, reduced the incidence of RV pulsus alternans (52% versus 20%, respectively; P = 0.056).

Figure 4.

Summary results and representative examples of Western immunoblots probed for talin (top), α-actinin (middle), and vinculin (below) in RV myocardium obtained at the end of RVPO in pigs treated with vehicle (V) or the calpain inhibitor MDL-28170 (M). Band intensities of talin, α-actinin, vinculin, and α-tubulin were each normalized to the mean of a fiduciary set of four samples that were repeated on each gel; α-tubulin was used as a loading standard. The talin/α-tubulin ratio was 21% higher in pigs treated with MDL-28170 than in pigs treated with vehicle (P = 0.039). A trend toward a higher α-actinin/α-tubulin ratio (9%) in MDL-28170 pigs was evident, but this difference did not reach statistical significance (P = 0.12). No difference in vinculin/α-tubulin ratio was evident between the two groups.

Figure 5.

Function versus talin: relationship between talin abundance and preservation of regional external RV work at the end of RVPO. The preservation of regional external RV work was strongly dependent on the preservation of talin abundance (r = 0.58, P = 0.0039). A similar relationship existed between talin/α-tubulin ratio and global RV stroke work (r = 0.47, P = 0.026). The large bold symbols (dots for vehicle, and X for MDL-28170) show the mean values (centroids) for vehicle and MDL-28170 pigs, respectively.

Figure 6.

Talin. Representative photomicrographs were obtained from 16-μm transverse sections of the RV free wall in pigs before (Baseline) and after 2 hours of acute RVPO immunostained for talin (red) and with (2-(4-amidinophenyl)-1H-indole-6-carboxamidine) (DAPI) (in blue, for nuclei). The second biopsy was obtained from a location at least 2 cm away from the first biopsy, and in a more proximal perfusion territory, to avoid sampling a region potentially affected by the first biopsy. Under baseline conditions, the talin signal was largely localized to the cell surface. After 2 hours of acute RVPO, patchy loss of the regular signal (white arrows) and areas of blurring (green arrows) were evident. RV myocardium from pigs identically instrumented and subjected to 2 hours of anesthesia without RV pressure overload (Sham) did not exhibit any significant differences from baseline biopsies, indicating that 2 hours of anesthesia without RVPO did not cause changes in talin organization according to immunohistology. Biopsies from pigs subjected to 2 hours of RVPO but pretreated with MDL-28170 (MDL) exhibited structures more like those of Baseline and Sham than RVPO. Image sets were obtained on a Leica digital deconvolution microscope (Leica Microsystems, Buffalo Grove, IL), using a ×63 oil immersion objective (NA 1.4) with 0.5-μm z-dimension steps, and processed using constrained iterative deconvolution. Image intensity and contrast were equalized among all images. Scale bar = 20 μm.

Figure 7.

Talin. Detail of a three-dimensional reconstruction of a 16-μm section obtained under baseline conditions (left) and after 2 hours of acute RVPO (right), immunostained for talin (red) and with DAPI (in blue, for nuclei). Under baseline conditions, talin exhibited a regular rib-like structure coaxial with the long axis of the myocyte (yellow arrow, left). After 2 hours of acute RVPO, disruption of the regular rib-like appearance of talin occurred (yellow arrow, right), with patchy areas of signal loss (white arrow) and blurring (green arrow). The imaging technique was described in Figure 5. Scale bar and grid = 10 μm.