Simultaneous administration of insulin-like growth factor-1 and darbepoetin alfa protects the rat myocardium against myocardial infarction and enhances angiogenesis

Abstract

Recent studies have shown that insulin growth factor-1 (IGF-1) and either erythropoietin (EPO) or the long-acting EPO analog Darbepoetin alfa (DA) protect the heart against ischemia/reperfusion (I/R) and myocardial infarction (MI). The present study examined the cardioprotective effect of simultaneous treatments with IGF-1 and DA in these models of cardiac injury. Rats were subjected to I/R or MI and were treated with IGF-1, DA, and a combination of IGF-1 and DA, or vehicle treatment. IGF-1 and DA treatments imparted similar protective effect by reducing infarct size. Moreover, these treatments led to improvement of cardiac function after I/R or MI compared to vehicle. In the reperfused heart, apoptosis was reduced with either or both IGF-1 and DA treatments as measured by reduced TUNEL staining and caspase-3 activity. In addition, after MI, treatment with IGF-1 or DA significantly induced angiogenesis. This angiogenic effect was enhanced significantly when IGF-1 and DA were given simultaneously compared to vehicle or either agents alone. These data indicate simultaneous pharmacological treatments with IGF-1 and DA protect the heart against I/R and MI injuries. This protection results in reduced infarct size and improved cardiac function. Moreover, this treatment reduces apoptosis and enhances angiogenesis in the ischemic heart.

Figure 1

Cardiac hemodynamics function at 72 hours of reperfusion. Rats were subjected to sham operation (sham) or 30 minutes ischemia followed by 72 hours of reperfusion and treated with vehicle, IGF‐1, DA, or IGF 1 + DA at the onset of ischemia. (A) LV + dP/dtmax, (B) LV − dP/dtmin, (C) heart rate, and (D) LVEDP. Cardiac function was recorded at baseline (B) and upon isoproterenol (0.1–1,000 ng) administration. (n= 8–10/group; *p < 0.05 vs. vehicle, ^# p < 0.05 vs. sham, ^§ p < 0.05 vs. DA and IGF‐1 alone.)

Figure 2

Post‐I/R infarction size. (A) Representative photographs of TTC‐stained rat heart sections from the different treatment groups. (B) LV infarct size expressed as percentage of the area at risk (AR; ischemic) in each group. (C) LV AR expressed as percentage of the total LV. (n= 5–10/group; *p < 0.05 vs. vehicle or sham; S, sham, V, vehicle.)

Figure 3

Post‐I/R apoptosis. (A) Representative photographs of TUNEL‐stained tissue sections from the different treatment groups. TUNEL (green), apoptotic nuclei, DAPI (blue), total nuclei, and TnI (red), myocytes. (B) TUNEL‐positive nuclei quantification represented as number per high‐power field (HPF). (C) Caspase‐3 activity represented as pmol of substrate (pNA) per mg of protein (mg p). (n= 5–15/group; **p < 0.01 or ***p < 0.001 vs. vehicle, ^# p < 0.05 vs. sham; S, Sham, V, vehicle.)

Figure 4

Cardiac echocardiographic function at 4 weeks of coronary occlusion. Rats were subjected to sham operation (sham) or 4 weeks of myocardial ischemia and treated with vehicle, IGF‐1, DA, or IGF‐1 + DA at the onset of ischemia. (A) Representative echocardiogram for sham (a), vehicle (b), IGF‐1 (c), DA (d), and IGF‐1 + DA (e) groups. (B) LV ejection fraction (LVEF). (C) LV fractional shortening (LVFS). Cardiac function was recorded at 4 weeks of myocardial ischemia. (n= 6–13/group; *p < 0.01 vs. vehicle, ^# p < 0.01 vs. sham.)

Figure 5

Cardiac hemodynamics function at 4 weeks of coronary occlusion. Rats were subjected to sham operation (sham) or 4 weeks of myocardial ischemia and treated with vehicle, IGF‐1, DA, or IGF‐1 + DA at the onset of ischemia. (A) LV + dP/dt _max, (B) LV − dP/dt _min, (C) heart rate, and (D) LVEDP. Cardiac function was recorded at baseline (B) and upon isoproterenol (0.1–1,000 ng) administration. (n= 8–10/group; *p < 0.05 vs. vehicle, ^# p < 0.05 vs. sham.)

Figure 6

Postischemia infarction size. (A) Representative photographs of TTC‐stained rat heart sections from the different treatment groups. (B) LV infarct size expressed as percentage of the LV in each group. (C) Heart weight (HW)‐to‐body weight (BW) ratio. (D) Lung weight (LW)‐to‐body weight (BW) ratio. (n= 4–22/group; *p < 0.05 vs. vehicle, ^# p < 0.05 vs. sham; S, sham, V, vehicle.)

Figure 7

Vessel density quantification. (A) Representative photographs of von Willbrand factor (vWF)‐stained tissue sections from sham (a), vehicle (b), IGF‐1 (c), DA (d), and IGF‐1 + DA (e) groups as well as a negative control (f). The vWF is stained in brown with a counterstaining with hematoxylin in purple. (B) Quantification of vessels in the infarct border zone. (C) Quantification of vessels in the remote area (septal wall). (n= 5–8/group; *p < 0.05 vs. vehicle, ^# p < 0.05 vs. sham, ^§ p < 0.05 vs. other pharmacological treatments; S, sham, V, vehicle, HPF, high power field.)

Figure 8

Hematocrit content. (A) Whole blood hematocrit content measured at 72 hours of reperfusion. (B) Whole blood hematocrit content measured at 4 weeks of myocardial ischemia. (n= 4–20/group; *p < 0.05 vs. vehicle, sham, and IGF‐1; S, sham, V, vehicle.)