Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

Background: The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Objectives: Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Methods: Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. Results: IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Conclusions: Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

Data demonstrating the anti-oxidant role of hemopexin in the heart.

The data presented in this article are related to the research article entitled Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress (G. Ingoglia, C. M. Sag, N. Rex, L. De Franceschi, F. Vinchi, J. Cimino, S. Petrillo, S. Wagner, K. Kreitmeier, L. Silengo, F. Altruda, L. S. Maier, E. Hirsch, A. Ghigo and E. Tolosano, 2017) [1]. Data show that heme induces reactive oxygen species (ROS) production in primary cardiomyocytes. H9c2 myoblastic cells treated with heme bound to human Hemopexin (Hx) are protected from heme accumulation and oxidative stress. Similarly, the heme-driven oxidative response is reduced in primary cardiomyocytes treated with Hx-heme compared to heme alone. Our in vivo data show that mouse models of hemolytic disorders, ß-thalassemic mice and phenylhydrazine-treated mice, have low serum Hx associated to enhanced expression of heme- and oxidative stress responsive genes in the heart. Hx-/- mice do not show signs of heart fibrosis or overt inflammation. For interpretation and discussion of these data, refer to the research article referenced above.

Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress.

Heart failure is a leading cause of morbidity and mortality in patients affected by different disorders associated to intravascular hemolysis. The leading factor is the presence of pathologic amount of pro-oxidant free heme in the bloodstream, due to the exhaustion of the natural heme scavenger Hemopexin (Hx). Here, we evaluated whether free heme directly affects cardiac function, and tested the therapeutic potential of replenishing serum Hx for increasing serum heme buffering capacity. The effect of heme on cardiac function was assessed in vitro, on primary cardiomyocytes and H9c2 myoblast cell line, and in vivo, in Hx-/- mice and in genetic and acquired mouse models of intravascular hemolysis. Purified Hx or anti-oxidants N-Acetyl-L-cysteine and α-tocopherol were used to counteract heme cardiotoxicity. In mice, Hx loss/depletion resulted in heme accumulation and enhanced reactive oxygen species (ROS) production in the heart, which ultimately led to severe systolic dysfunction. Similarly, high ROS reduced systolic Ca2+ transient amplitudes and fractional shortening in primary cardiomyocytes exposed to free heme. In keeping with these Ca2+ handling alterations, oxidation and CaMKII-dependent phosphorylation of Ryanodine Receptor 2 were higher in Hx-/- hearts than in controls. Administration of anti-oxidants prevented systolic failure both in vitro and in vivo. Intriguingly, Hx rescued contraction defects of heme-treated cardiomyocytes and preserved cardiac function in hemolytic mice. We show that heme-mediated oxidative stress perturbs cardiac Ca2+ homeostasis and promotes contractile dysfunction. Scavenging heme, Hx counteracts cardiac heme toxicity and preserves left ventricular function. Our data generate the rationale to consider the therapeutic use of Hx to limit the cardiotoxicity of free heme in hemolytic disorders.