Protein Kinase C Inhibition With Ruboxistaurin Increases Contractility and Reduces Heart Size in a Swine Model of Heart Failure With Reduced Ejection Fraction

Figures

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  Figure 1. Study Timeline Protocol used to induce acute myocardial infarction (MI) and functionally assess the progression of pathological remodeling. Timeline for delivery of dobutamine (DOB) and ruboxistaurin (RBX) at 3 months’ post-MI. LAD = left anterior descending coronary artery.

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  Figure 2. Gross, Cellular, and Molecular Remodeling 3 Months PMI (A) Gross images of sham heart, whole organ (left), and cross section (right) of mid-myocardium. (B) Gross images of 12-week PMI heart, whole organ (left), and cross section of mid-myocardium (right). (C) Scar size analysis showing viable (white) and scar tissue (black) as a percentage of total myocardial volume at 12 weeks PMI. (D) Bright-field images were taken of Masson’s trichrome–stained mid-myocardial cross sections of the infarct zone (IZ), border zone (BZ), and remote zone (RZ) of a 3 months PMI animal. The 10× (outside images) and 40× (interior images) were acquired to demonstrate fibrosis and the deposition of collagen in the AW and LW of the PMI animal. (E) Heart weight-to-body weight (HW/BW) ratio. (F) Myocyte cross-sectional area calculated from the 2 viable zones (border zone [BZ] and remote zone [RZ]) in sham animals versus 3 months PMI animals. The images represent the BZ in the sham group (F1) and the 3 months PMI group (F2). Large image is 20× and small image (top right corner) is 40×. Data are shown as the mean by anatomic region in the graphs below. (G) Animals underwent sham (n = 6) or MI (n = 6)…

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  Figure 3. LV Remodeling Echocardiography was performed before, 1 month (Mo), 2Mo, and 3Mo post-myocardial infarction (PMI). (A) Left ventricular (LV) end-diastolic volume (LVEDV), (B) end-systolic volume (LVESV), and (C) ejection fraction (LVEF) images were acquired using the 2-dimensional parasternal long-axis view. Calculation was performed using the Simpson method. Open bars represent before MI, red bars represent PMI. *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.005; ****p ≤ 0.005 versus baseline.

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  Figure 4. Hemodynamic Deterioration due to Acute MI Invasive hemodynamics were performed at baseline and 3 months PMI. (A) Representative pressure-volume loops from before and 3 months after MI. (B) Both end-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure volume relationship (EDPVR) are shown. ESPVR and EDPVR are shifted rightward at 3 months PMI versus baseline. (C) The pre-load recruitable stroke work (PRSW), a load-independent measure. is significantly reduced after MI. (D) LV volume at pressure of 10 mm Hg (LVVPed10) was significantly increased post MI, indicating an increase in capacitance. (E) LVVPes80 was also significantly increased, indicating a reduction in contractility as it pertains to the fore of contraction. (F) Left ventricular ejection fraction was reduced, indicating dilation at 3 months after MI. ***p ≤ 0.001, ****p ≤ 0.0005 versus baseline (B). Abbreviations as in Figure 2.

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  Figure 5. Altered Hemodynamic Response to DOB 3 Months PMI Invasive hemodynamics were performed at baseline ± DOB and 3 months PMI ± DOB. (A) Representative pressure-volume loops in the presence and absence of DOB at baseline and 3 months after MI. (B) EDPVR was not altered at either time point ± DOB. (C) ESPVR was significantly altered (specifically the Ees) at baseline ± DOB, but not at 3 months PMI. (D) PRSW was increased at both time points. (E) LVVPed10 was not significantly reduced in the presences of DOB at 3 months PMI. (F) The LVVPes80 was significantly reduced at baseline ± DOB, but not 3 months PMI ± DOB. (G) EF was increased significantly by DOB at 3 months PMI, but only due to modest reduction in ESV. (H) The Ees, 1 of 2 determinants of the ESPVR, was significantly increased at baseline + DOB, but not at 3 months PMI. (I) The volume intercept, second determinant of the ESPVR, was not significantly changed at 3 months PMI ± DOB. Open bars = baseline (B), and gray bars = dobutamine (+), red bars = 3Mo PMI. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001, ****p ≤ 0.0005 versus baseline; †p ≤ 0.05 versus 3 months PMI; #p ≤ 0.05, ###p ≤ 0.001, ####p ≤ 0.0005 versus baseline + DOB. NS =…

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  Figure 6. Assessment of LV Volumes With RBX Treatment After MI and in Control Animals Echocardiography was performed before and after RBX treatment at 3 months PMI. (A) Left ventricular (LV) end-diastolic volume (LVEDV) was reduced + RBX. (B) LV end-systolic volume (LVESV) was reduced + RBX. (C) LV ejection fraction (LVEF) was increased + RBX. In age-matched controls (CON), ± RBX had no effect on (D) LVEDV, (E) LVESV, or (F) LVEF. Images were acquired using the 2-dimensional parasternal long-axis view. Calculation was performed using the Simpson method. †p ≤ 0.05, ††p ≤ 0.005 versus 3 months PMI. Abbreviations as in Figures 1, 2, and 4.

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  Figure 7. RBX Reduces EDPVR and Improves Contractility Serial invasive hemodynamics were performed at baseline (B), 3 months PMI ± DOB, and ± RBX. (A) Representative pressure-volume loops at 3 months after MI + DOB or + RBX. (B) EDPVR was not altered ± DOB, but shifted leftward with RBX. (C) ESPVR was not significantly altered 3 months PMI + DOB but was also shifted leftward with RBX. (D) PRSW was increased at both time points but to a greater extend with RBX. (E) LVVPed10 was significantly reduced in the presence of RBX at 3 months PMI versus 3 months PMI +DOB. (F) The LVVPes80 was significantly reduced at 3 months PMI +RBX, but not ± DOB. (G) EF was increased significantly by DOB and RBX at 3 months PMI, but due to profound reductions in the EDV with RBX. (H) The Ees, 1 of 2 determinants of the ESPVR, was significantly increased at +RBX versus 3 months PMI. (I) The volume intercept, second determinant of the ESPVR, was significantly changed at 3 months PMI + RBX vs. ± DOB. RBX (yellow bars = 2.5 μg/kg/min RBX. *p ≤ 0.05, **p ≤ 0.005 versus baseline; †p ≤ 0.05, ††††p ≤ 0.0005 versus 3 months PMI; ‡p ≤ 0.05, ‡‡‡p ≤ 0.001, ‡‡‡‡p ≤ 0.0005 versus 3 months PMI + DOB. Abbreviations as in…

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  Figure 8. RBX Reduces PKCα Phosphorylation at T638 and Does Not Directly Alter PKA-Dependent Signaling (A) Western blot for total PKCα and phosphorylation at threonine 638 (T638) after MI ± RBX. T638 phosphorylation was reduced in the presences of RBX (B) Correlation of T638 phosphorylation and echocardiographic volumetric analysis. Increased phosphorylation was positively correlated to increases in LVEDV and LVESV at 3 months PMI, and reduced LVEF. (C) Western blot for total phospholamban (PLN) and phosphorylation sites, serine 16 (S16) and threonine 17 (T17). RBX had no effect on the S16 or T17 phosphorylation. †p ≤ 0.05 versus 3 months PMI. Abbreviations as in Figures 1, 2, 4, and 5.