Heart failure-associated anemia: bone marrow dysfunction and response to erythropoietin

Abstract

Heart failure (HF)-associated anemia is common and has a poor outcome. Because bone marrow (BM) dysfunction may contribute to HF-associated anemia, we first investigated mechanisms of BM dysfunction in an established model of HF, the transgenic REN2 rat, which is characterized by severe hypertrophy and ventricular dilatation and SD rats as controls. Secondly, we investigated whether stimulation of hematopoiesis with erythropoietin (EPO) could restore anemia and BM dysfunction. After sacrifice, erythropoietic precursors (BFU-E) were isolated from the BM and cultured for 10 days. BFU-E were quantified and transcript abundance of genes involved in erythropoiesis were assayed. Number of BFU-E were severely decreased in BM of REN2 rats compared to SD rats (50 ± 6.2 vs. 6.4 ± 1.7, p < 0.01). EPO treatment increased hematocrit in the SD-EPO group (after 6 weeks, 49 ± 1 vs. 58 ± 1%, p < 0.01); however, in the mildly anemic REN2 rats, there was no effect (43 ± 1 vs. 44 ± 1%). This was paralleled by a 67% decrease in BFU-E in BM of REN2 rats compared to SD (p < 0.01). EPO significantly improved BFU-E in both SD and REN2 but could not restore this to control levels in the REN2 rats. Expression of several genes involved in differentiation (LMO2), mobilization (SDF-1), and iron incorporation (transferrin receptor) of the BM were differentially expressed in REN2 rats compared to SD rats, and EPO did not normalize this. Altogether, these results suggest that BM dysfunction is an important contributor to HF-associated anemia and that EPO is not an effective agent to treat HF-associated anemia.

Fig. 1

Overview of different factors involved in differentiation and mobilization during different stages of erythropoiesis, which expression was measured (results shown in Fig. 5). Adapted from Koury et al. [12], according to Hanson et al. [29], Ohneda et al. [31], and Jin et al. [32]. Factors highlighted in green are positive regulators, factor highlighted in red is a negative regulator. HSC hematopoietic stem cell, BFU-E burst forming unit-erythroid, CFU-E colony forming unit-erythroid, RET reticulocyte, RBC red blood cell, LMO2 LIM domain only 2 protein, TAL T cell acute lymphocytic leukemia protein, GATA GATA binding protein, FOG friend of GATA, MMP matrix metallopeptidase, SDF stromal cell-derived factor

Fig. 2

Shows that the used animal model is a model of hypertensive heart failure. a Mean arterial pressure (MAP) during the experiment. MAP is significantly higher at all time points for REN2 groups compared to SD groups. b Heart weight normalized to tibia length. c Left ventricular end diastolic pressure (LVEDP) at sacrifice. d mRNA expression of atrial natriuretic peptide (ANP) in left ventricular tissue. ANP is expressed as fold change. HW heart weight, Base baseline, Wk week, PL placebo. *p < 0.05; **p < 0.01

Fig. 3

a Change in hematocrit (Ht) levels during experiment. EPO administration cannot increase Ht levels in REN2 rats. b Number of burst forming units-erythroid (BFU-E, normalized to SD-PL). c Number of colony forming units (CFU, normalized to SD-PL). PL placebo, BM bone marrow, Wk week, #p < 0.05 vs. SD-PL, REN2-PL and REN2-EPO, ¶p < 0.05 vs. REN2-PL and REN2-EPO, *p < 0.05, **p < 0.01

Fig. 4

a–d Typical examples of FACS analysis. Every dot is a single cell count. The x-axis (Comp-APC-A) is the gate for fluorescent cells, the y-axis (SSC-A) is the cell size. Red color is high density of cell count; blue color is lower density of cell count. e Number of the erythroid c-kit⁺ cells in bone marrow. PL placebo, BM bone marrow. *p < 0.05; **p < 0.01

Fig. 5

mRNA expression of different factors involved in differentiation (a, b) and mobilization (c, d) of c-kit⁺ cells. mRNA expression of different factors involved in iron metabolism (e, f). LMO2 LIM domain only 2 protein, GATA-1 GATA binding protein, MMP9 matrix metallopeptidase, SDF-1 stromal cell-derived factor, PL placebo. *p < 0.05; **p < 0.01

Fig. 6

Schematic overview of the mechanisms of bone marrow dysfunction involved in HF-associated anemia. Different triggers of anemia in heart failure such as insensitivity of EPO, iron deficiency, and neurohormonal activation have a negative effect on the hematopoietic stem cells in the bone marrow. Furthermore, impaired proliferation, differentiation, mobilization, and iron incorporation in hematopoietic stem cells contribute to the bone marrow dysfunction, causing anemia and adding to the heart failure syndrome, which in turn triggers anemia in heart failure, etc. EPO treatment does not rescue the dysfunction of the erythroid lineage and does not improve heart failure-associated anemia. SNS sympathetic nervous system, RAS renin–angiotensin system