Functional impairment of human resident cardiac stem cells by the cardiotoxic antineoplastic agent trastuzumab

Abstract

Trastuzumab (TZM), a monoclonal antibody against the ERBB2 protein, increases survival in ERBB2-positive breast cancer patients. Its clinical use, however, is limited by cardiotoxicity. We sought to evaluate whether TZM cardiotoxicity involves inhibition of human adult cardiac-derived stem cells, in addition to previously reported direct adverse effects on cardiomyocytes. To test this idea, we exposed human cardiosphere-derived cells (hCDCs), a natural mixture of cardiac stem cells and supporting cells that has been shown to exert potent regenerative effects, to TZM and tested the effects in vitro and in vivo. We found that ERBB2 mRNA and protein are expressed in hCDCs at levels comparable to those in human myocardium. Although clinically relevant concentrations of TZM had no effect on proliferation, apoptosis, or size of the c-kit-positive hCDC subpopulation, in vitro assays demonstrated diminished potential for cardiogenic differentiation and impaired ability to form microvascular networks in TZM-treated cells. The functional benefit of hCDCs injected into the border zone of acutely infarcted mouse hearts was abrogated by TZM: infarcted animals treated with TZM + hCDCs had a lower ejection fraction, thinner infarct scar, and reduced capillary density in the infarct border zone compared with animals that received hCDCs alone (n = 12 per group). Collectively, these results indicate that TZM inhibits the cardiomyogenic and angiogenic capacities of hCDCs in vitro and abrogates the morphological and functional benefits of hCDC transplantation in vivo. Thus, TZM impairs the function of human resident cardiac stem cells, potentially contributing to TZM cardiotoxicity.

Figure 1.

ERBB2 mRNA and protein expression in human CDCs. (A): Reverse transcription-polymerase chain reaction shows expression of ERBB isoforms in human myocardium (RV and RA both obtained from a nonfailing human control heart) and hCDCs isolated from three different patients. 18S RNA served as a control. (B): Representative analysis of ERBB2 protein quantified by flow cytometry (20 ± 3% of hCDCs expressed ERBB2 protein, n = 4). The breast cancer cell line MCF-7, known to express ERBB2, served as positive control. (C): In vitro proliferation of human cardiosphere-derived cells (hCDCs) was not affected by TZM. No statistically significant differences were seen for proliferation of freshly isolated hCDCs (days 1–8) versus hCDCs that were first incubated for up to 13 days with TZM, and proliferation on days 14–21 was then measured using the WST-8 assay (at least n = 4 different hCDCs isolates for each condition). (D): TZM did not change the percentage of apoptotic or c-kit-positive hCDCs (n = 5 different human CDCs). Abbreviations: hCDC, human cardiosphere-derived cell; NS, not significant; RA, right atrium; RV, right ventricle; TZM, trastuzumab.

Figure 2.

In vitro angiogenesis assay. Endothelial markers CD31 and CD34 were expressed at the mRNA level (A) and protein level (B). In summary, 15 ± 7% and 7 ± 2% of hCDCs expressed CD31 and CD34 protein, respectively; n = 6. (C): Tube formation on Matrigel angiogenesis assay was similar in hCDCs (top) and HUVECs (bottom). Magnification, ×10. (D): Representative images of a time-course experiment. Early angiogenic response after 4 hours was highlighted by formation of vascular tubes giving rise to a complex mesh-like structure. After 24 hours, cells formed a central cluster in the middle of the well (top right). Several days later, cells started migrating out from the central cell cluster, gradually covering the entire well over the next 2–3 weeks (bottom). Magnification, ×4 (top) and ×10 (bottom). Abbreviations: d, days; h, hours; hCDCs, human cardiosphere-derived cells; HUVEC, human umbilical vein endothelial cells; RA, right atrium; RV, right ventricle.

Figure 3.

Effect of TZM on in vitro angiogenesis. (A): The size and complexity of the microvascular network on Matrigel matrix after 17 days in culture differed significantly between control and TZM-treated hCDCs. (B): Continued incubation with TZM-containing medium slowed the growth and reduced the complexity of the microvascular network by >50% (n = 8 different human CDCs, each plated and analyzed in triplicate for control and TZM groups). Abbreviations: a.u., arbitrary units; hCDC, human cardiosphere-derived cells; TZM, trastuzumab.

Figure 4.

In vitro assay for early cardiogenic differentiation. CDCs were transduced with lentiviral vectors expressing firefly luciferase under the transcriptional control of the cardiac-specific promoter of the sodium-calcium exchanger. Subsequently, hCDCs were incubated for 11 days with control medium, medium + DMSO, or medium with either of the two cardiogenic sulfonyl hydrazone compounds, JL-265-010 and JL-265-037. Then, luciferase activity was determined with in vitro luciferase assays, and results were normalized to cell number. (A): Cardiogenic differentiation was significantly inhibited by TZM compared with control (n = 5). (B): Quantitative real-time reverse transcription-polymerase chain reaction showed expression of cardiac-specific mRNAs, including NPPB, TPMA, TNNI3, and ATP2A2, to be twofold higher in the control versus the TZM group (n = 5, p < .05 for all comparisons). Abbreviations: ATP2A2, SERCA2; DMSO, dimethyl sulfoxide; NPPB, pro-brain natriuretic peptide; RLU, relative light units; TNNI3, cardiac troponin I; TPMA, tropomyosin; TZM, trastuzumab.

Figure 5.

In vivo effect of trastuzumab. (A): Biweekly intraperitoneal injection of TZM for 3 weeks did not lead to left ventricular dysfunction in SCID-beige mice. (B): Biweekly intraperitoneal administration of TZM abrogated the functional benefit of hCDCs injected into the border zone of acutely infarcted murine left ventricular myocardium. Injection of hCDCs alone led to a 10% increase in ejection fraction compared with animals that received hCDCs and TZM. Of note, there was no difference between the treatment group (hCDCs + TZM) and the two negative control groups included in the study, that is, injection of human dermal fibroblasts and injection of PBS. (C): Differences in postinfarct remodeling were evident by a thinner infarct scar in animals receiving TZM, whereas the difference in infarct area between the two groups was not statistically significant (the numbers of animals are indicated on the bars). Abbreviations: EF, ejection fraction; hCDC, human cardiosphere-derived cells; NS, not significant; PBS, phosphate-buffered saline; TZM, trastuzumab.

Figure 6.

In vivo effect on angiogenesis. (A): Representative images of the infarct border zone myocardium of hCDCs + PBS-treated (top) and hCDCs + TZM-treated (bottom) animals, demonstrating a reduced number of capillaries in TZM-treated animals. Capillaries were stained with isolectin B4-fluorescein (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue) and with an antibody targeting human nuclear antigen (red). (B): After capillaries were stained with isolectin B4-fluorescein, green pixels were normalized to the number of total nuclei in the imaged infarct border zone area. Similar results were obtained when green pixels were divided by the number of human nuclei, stained with an antibody targeting the human nuclear antigen (Chemicon; data not shown). Images were taken at ×20 magnification. Scale bars = 50 μm. Abbreviations: hCDC, human cardiosphere-derived cells; PBS, phosphate-buffered saline; TZM, trastuzumab.