Cardiomyocyte PDGFR-β signaling is an essential component of the mouse cardiac response to load-induced stress

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-β expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-β signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-β was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-β is a regulator of the compensatory cardiac response to pressure overload–induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

PDGF receptors and/or ligands are overexpressed in a wide variety of malignancies (1–4). In the past decade, a number of inhibitors of receptor tyrosine kinases (RTKs) whose targets include PDGFR have emerged as agents with remarkable efficacy in the treatment of human cancers (5–7). However, several of these agents have been linked to the development of cardiomyopathy in subsets of treated patients.

Recent reports indicate that the anticancer agent sunitinib, whose targets include PDGFR, causes cardiac dysfunction in up to 19% of treated patients (8), and we and others have reported the development of clinical heart failure in subsets of sunitinib-treated patients (8–10). Sorafenib, another RTK inhibitor that targets PDGFR, was recently associated with myocardial damage and cardiac dysfunction in renal cell carcinoma patients (10). In addition, imatinib, whose targets include PDGFR in addition to Abl kinase, has been linked to the development of cardiomyopathy (11), although this appears to be an infrequent clinical occurrence (12). Importantly, both sunitinib and sorafenib lead to the development of subacute, severe hypertension in substantial numbers of patients (8, 13), while imatinib rarely causes hypertension.

Linking the incidence of cardiomyopathy associated with anticancer PDGFR inhibitors with the relative incidences of hypertension associated with these drugs led us to hypothesize that PDGFR signaling in the heart may play a key role in the cardiac response to vascular stress such as hypertension. However, little is known regarding the role of PDGFR signaling in adult cardiomyocytes. We show here that cardiomyocyte PDGFR-β expression and activation rise dramatically in response to pressure overload–induced stress. Furthermore, we show that knockout of PDGFR-β in the cardiomyocyte results in cardiac dysfunction, heart failure, and a marked defect in stress-induced cardiac angiogenesis. Mechanistically, we demonstrate that PDGFR-β is an essential regulator of the paracrine angiogenic capacity, or angiogenic potential, of cardiomyocytes.

PDGFR-β expression in cardiomyocytes increases dramatically in response to pressure overload–induced stress. We exposed C57BL/6 mice to transverse aortic constriction (TAC), in which a ligature is placed at the aortic arch, resulting in acute pressure overload. As expected, we observed profound increases in cardiac weight (Figure 1A) and cardiac thickness (Figure 1B) in mice exposed to TAC. Cardiac lysates from mice exposed to TAC exhibited increased levels of PDGFR-β persisting to 14 days after TAC (Figure 1C). Quantitative densitometry revealed a 10-fold increase in PDGFR-β protein levels in the heart 7 days after TAC (Figure 1D). Enhanced PDGFR-β expression after TAC was predominantly seen on cardiomyocytes (Figure 1E), as opposed to non-myocyte cells within the heart such as fibroblasts or endothelial cells. A substantial increase in phosphorylated, activated PDGFR-β was seen in the heart 7 days after TAC (Figure 1F) and 14 days after TAC (data not shown), suggesting that changes in PDGFR-β in the cardiomyocyte after TAC were functionally important. Marked increases in levels of phosphorylated Akt and ERK1/2, established downstream effectors of PDGFR-β, were also observed in the heart after TAC. Immunohistochemistry with a phospho-specific PDGFR-β antibody demonstrated that phospho–PDGFR-β after TAC was increased predominantly in cardiomyocytes (Figure 1G). Levels of PDGFR-α were increased in the hearts of mice exposed to TAC compared with sham-operated mice (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI39434DS1), but we observed no increase in phosphorylation of PDGFR-α (data not shown), suggesting a dominant biological role of PDGFR-β compared with PDGFR-α in the heart after exposure to load stress. We also observed significantly increased levels of local expression of the PDGFR-β ligands PDGF-B and PDGF-C 3 days after TAC (Supplemental Figure 2), while levels of PDGF-A and PDGF-D were significantly decreased in the heart 3 days after TAC, suggesting that dynamic changes in local PDGF expression may contribute to activation of PDGFR-β after TAC.

Figure 1

PDGFR-β expression and phosphorylation in cardiomyocytes increase dramatically in response to pressure overload stress. (A) Heart weight/body weight ratios in 8- to 12-week-old C57BL/6 mice exposed to TAC or sham surgery (Sham). (B) Ratio of the posterior wall thickness of hearts from mice exposed to TAC versus sham surgery. Day 0 indicates baseline levels, prior to surgery. (C) Western blot of PDGFR-β levels in cardiac lysates harvested after the indicated number of days from animals exposed to TAC or sham surgery. (D) Ratio of PDGFR-β levels (normalized for GAPDH) from cardiac lysates from animals exposed to TAC versus sham surgery at the indicated time points (n = 4 samples in each group). (E) Immunohistochemical staining for PDGFR-β in cardiac sections from mice exposed to sham surgery (upper panel) or TAC for 7 days. Arrows indicate cardiomyocytes intensely stained for PDGFR-β. Results are representative of at least 3 separate, independent cardiac samples. Scale bars: 50 μm. (F) Western blot probed for phospho-/total levels of PDGFR-β, Akt, and ERK1/2 in hearts of unoperated mice (Control) or mice 7 days after TAC or sham surgery. Results are representative of 4 samples in each group. (G) Immunohistochemical staining for phospho–PDGFR-β in cardiac sections from mice exposed to sham surgery (upper panel) or TAC for 7 days. Arrows indicate cardiomyocytes intensely stained for phospho–PDGFR-β. Scale bars: 50 μm. Asterisks indicate that statistical comparison was between TAC and sham surgery at the specified time point. P values were determined by unpaired, 2-tailed Student’s t test.

Cardiomyocyte PDGFR-β is not required for normal cardiac development or basal adult cardiac function. To further explore the role of PDGFR-β signaling in cardiac function, we constructed mice in which PDGFR-β was knocked out in cardiomyocytes. This was done by crossing mice expressing the Cre recombinase driven by the Nkx2.5 promoter (Nkx2.5-Cre mice) (14) with mice in which PDGFR-β was flanked by loxP sites (Pdgfrb^fl/fl mice) (15). Nkx2.5 is a cardiac-specific transcription factor that exhibits expression at high levels in the heart beginning at 9.5 dpc (14). Nkx2.5-Cre:Pdgfrb^fl/fl mice (cardiac-specific PDGFR-β–knockout mice; Pdgfrb^Nkx-Cre) were born at the expected Mendelian frequency (data not shown) and were able to support pregnancy. To confirm that the knockout strategy was successful and specific, we isolated genomic DNA from the hearts of newborn Pdgfrb^Nkx-Cre mice. The presence of the lower PCR band represents the Cre-excised allele (Figure 2A), and this was seen in the heart, but not in mouse tails or skeletal muscle, while the higher band, the nonexcised floxed allele, was markedly diminished in the heart compared with other tissues. Furthermore, representative Western blots from hearts of 4-week-old mice demonstrated reduced levels of PDGFR-β protein in Pdgfrb^Nkx-Cre compared with Pdgfrb^fl/fl controls (Figure 2B). Analysis of mutant embryos at 15.5 dpc revealed normal cardiac trabeculation and no evidence of septal or outflow tract abnormalities (data not shown), as observed in conventional knockout models of PDGF-B (16) and PDGFR-α (17).

Figure 2

Cardiac-specific PDGFR-β–knockout mice develop ventricular dilatation and heart failure in response to pressure overload. (A) PCR of genomic DNA from tails, skeletal muscle (SM), or hearts (Hrt) from newborn cardiac-specific PDGFR-β–knockout mice (Pdgfrb^Nkx-Cre) mice using primer pairs specific for floxed allele (upper band) or deleted allele (lower band). Results are representative of 3 independent experiments. (B) Western blot of cardiac lysates from 4-week-old Nkx2.5-Cre:Pdgfrb^fl/fl mice (Pdgfrb^Nkx-Cre) or non–Cre-expressing littermate control (Pdgfrb^fl/fl) mice probed with an antibody against PDGFR-β. (C) Ratio of right/left (R/L) peak carotid velocity 24 hours after 8- to 12-week-old Pdgfrb^Nkx-Cre or Pdgfrb^fl/fl mice were exposed to TAC (n = 8 in each group). (D) Cardiac ejection fraction of Pdgfrb^fl/fl mice or Pdgfrb^Nkx-Cre mice prior to TAC (day 0) or at various time points after TAC, as measured by MRI. (E) Heart weight/body weight ratios of Pdgfrb^fl/fl versus Pdgfrb^Nkx-Cre mice prior to TAC (day 0) or 14 days after TAC. (F) Representative short-axis MRI still frame images taken at the mid-ventricular level in Pdgfrb^fl/fl or Pdgfrb^Nkx-Cre mice prior to TAC exposure (left panels) or 14 days after TAC exposure (right panels). (G) Ratio of lung weight/body weight of Pdgfrb^fl/fl versus Pdgfrb^Nkx-Cre mice prior to TAC or 14 days after TAC. P values were determined by unpaired, 2-tailed Student’s t test. Numbers inside the bars indicate the number of animals analyzed.

When animals were 6 weeks of age, we observed no significant difference in cardiac ejection fraction in Pdgfrb^Nkx-Cre compared with Pdgfrb^fl/fl or Nkx2.5-Cre control mice (Table 1). Notably, we did observe a small but significant increase in heart weight/body weight ratio in Pdgfrb^Nkx-Cre compared with Pdgfrb^fl/fl or Nkx2.5-Cre control mice, driven largely by nonsignificant increases in the thickness of the intraventricular septum and posterior walls. In addition, we observed small, significant increases in lung weight/body weight ratios in Pdgfrb^Nkx-Cre mice. Importantly, cardiac ejection fractions, heart weights, and ventricular chamber sizes were normal in 8-month-old Pdgfrb^Nkx-Cre mice and were not different from those of Pdgfrb^fl/fl or Nkx2.5-Cre controls (data not shown). Thus, cardiomyocyte PDGFR-β signaling does not appear to play a key role in cardiac development or in basal adult cardiac function.

Table 1

Cardiac function and morphometry of 6-week-old cardiac-specific PDGFR-β–knockout mice (Pdgfrb^Nkx-Cre) compared with controls (Nkx2.5-Cre and Pdgfrb^fl/fl)

PDGFR-β cardiac-specific knockout mice develop ventricular dilatation and heart failure in response to pressure overload. To determine whether cardiomyocyte PDGFR-β is a required component of the cardiac response to pressure overload, we exposed 8- to 12-week-old Pdgfrb^Nkx-Cre mice or littermate Pdgfrb^fl/fl controls to TAC stress. The ratio of right-to-left carotid flow velocities, a surrogate of the degree of aortic impedance imposed in our TAC model (18), was similar in Pdgfrb^Nkx-Cre mice and Pdgfrb^fl/fl controls (Figure 2C). Notably, cardiac ejection fractions in Pdgfrb^Nkx-Cre mice were significantly decreased compared with those in Pdgfrb^fl/fl controls at 7 days after TAC, with a further decline 14 days after TAC (Figure 2D). In addition, Pdgfrb^Nkx-Cre mice displayed increased heart weight/body weight ratios 14 days after TAC (Figure 2E), indicative of enhanced pathologic hypertrophy in Pdgfrb^Nkx-Cre mice after TAC stress. Cardiomyocyte cross-sectional area 14 days after TAC was significantly increased in Pdgfrb^Nkx-Cre mice compared with Pdgfrb^fl/fl controls (Supplemental Figure 3), further indicative of a pathologic hypertrophy phenotype. Representative short-axis systolic and diastolic still frame MRI images (Figure 2F) demonstrated the acquired cardiac dysfunction seen in Pdgfrb^Nkx-Cre mice 14 days after TAC. Lung weights were significantly increased in Pdgfrb^Nkx-Cre mice 14 days after TAC (Figure 2G), indicative of pulmonary edema and functional heart failure.

Inducible, cardiac-specific PDGFR-β–knockout mice develop severe heart failure in response to TAC stress. Our findings in Pdgfrb^Nkx-Cre mice suggest that PDGFR-β signaling plays a key role in the cardiac response to load stress. However, we could not exclude the possibility that developmental abnormalities and/or expression of Cre recombinase in Pdgfrb^Nkx-Cre mice predispose these mice to cardiac failure upon exposure to TAC. To specifically determine the role of PDGFR-β signaling in the adult cardiac response to load-induced stress, we crossed Pdgfrb^fl/fl mice with mice expressing the MerCreMer transgene driven by the α-MHC promoter (MerCreMer mice), which exhibit high cardiac levels of nuclear Cre recombinase activity only in the presence of tamoxifen (19). Hearts from α-MHC-MerCreMer:Pdgfrb^fl/fl mice (inducible, cardiac-specific PDGFR-β–knockout mice, Pdgfrb^MerCre) expressed reduced levels of PDGFR-β (Figure 3A) compared with non–Cre-expressing, tamoxifen-treated Pdgfrb^fl/fl controls. We observed no compensatory differences in the expression of PDGFR-α in the hearts of Pdgfrb^MerCre mice compared with Pdgfrb^fl/fl control mice (Supplemental Figure 4). Inducible mutants were exposed to a similar degree of TAC stress as either Pdgfrb^fl/fl or MerCreMer control mice (Figure 3B). Heart weights of tamoxifen-treated Pdgfrb^MerCre mice significantly increased in response to TAC compared with those of tamoxifen-treated Pdgfrb^fl/fl or MerCreMer controls (Figure 3C). Cardiomyocyte cross-sectional area 14 days after TAC was significantly increased in Pdgfrb^MerCre mice compared with Pdgfrb^fl/fl and MerCreMer controls (Supplemental Figure 5), further indicative of a pathologic hypertrophy phenotype in Pdgfrb^MerCre mice. Pdgfrb^MerCre mice experienced significant declines in cardiac ejection fraction 7 days after TAC and severe cardiac dysfunction 14 days after TAC compared with Pdgfrb^fl/fl or MerCreMer controls (Figure 3D). Cardiac dysfunction and pathologic hypertrophy in Pdgfrb^MerCre mice was associated with increased ventricular dilatation, seen 14 days after TAC in Pdgfrb^MerCre mice (Figure 3E). Representative hearts from Pdgfrb^MerCre mice and Pdgfrb^fl/fl controls 14 days after TAC demonstrate the observed cardiac dilatation (Figure 3F). MRI cine videos demonstrate the load-induced dilated cardiomyopathy phenotype in Pdgfrb^MerCre mice (Supplemental Video 1). Significant increases in lung weight, indicative of pulmonary edema, were seen in Pdgfrb^MerCre mice compared with either MerCreMer or Pdgfrb^fl/fl control mice beginning at 7 days after TAC, and differences were even more striking 14 days after TAC (Figure 3G). Expression of B-type natriuretic peptide (BNP), a marker of pathologic hypertrophy and heart failure (20), was increased in hearts of Pdgfrb^MerCre mice as early as 3 days after exposure to TAC (Figure 3H).

Figure 3

Inducible, cardiac-specific PDGFR-β–knockout mice develop severe heart failure in response to pressure overload. (A) Expression of PDGFR-β in hearts from α-MHC-MerCreMer:Pdgfrb^fl/fl mice (inducible, cardiac-specific PDGFR-β–knockout mice, Pdgfrb^MerCre) or Pdgfrb^fl/fl controls harvested 7 days after exposure to tamoxifen. Results are representative of 4 independent hearts assessed from each group. (B) Ratio of right/left peak carotid velocity 24 hours after 8- to 12-week-old Pdgfrb^MerCre, MerCreMer, or Pdgfrb^fl/fl mice were exposed to TAC. All groups of mice were treated with tamoxifen in an identical manner prior to baseline analysis and exposure to TAC stress (see Methods). (C) Heart weight/body weight ratios in 8- to 12-week-old Pdgfrb^MerCre, MerCreMer, or Pdgfrb^fl/fl mice after TAC. (D) Cardiac ejection fraction of Pdgfrb^MerCre, MerCreMer, or Pdgfrb^fl/fl mice prior to TAC or at time points after TAC, as measured by MRI. (E) LV end diastolic volume of Pdgfrb^MerCre, MerCreMer, or Pdgfrb^fl/fl mice 14 days after TAC. (F) Representative hearts from Pdgfrb^fl/fl or Pdgfrb^MerCre mice 14 days after TAC. (G) Expression levels of BNP assessed from mRNA from hearts of Pdgfrb^fl/fl or Pdgfrb^MerCre mice prior to TAC or at time points after TAC (n = 4 samples per group at each time point). (H) Ratio of lung weight/body weight of Pdgfrb^MerCre, MerCreMer, or Pdgfrb^fl/fl mice at baseline or at time points after TAC. P values were determined by ANOVA, and significant differences between Pdgfrb^MerCre and control (MerCreMer and Pdgfrb^fl/fl) mice were confirmed with Tukey’s test. Numbers inside the bars indicate the number of animals analyzed.

Inducible cardiac-specific PDGFR-β–knockout mice exhibit impaired activation of Akt and MAPK pathways in the progression to heart failure. To gain insight into the mechanism of load-induced heart failure in Pdgfrb^MerCre mice, we measured changes in the PI3K and MAPK pathways, effectors of PDGFR signaling (21) known to mediate the compensatory cardiac response to load-induced stress (22). Levels of phosphorylation of p38 kinase, whose role in cardiac protection is ambiguous (23), were significantly decreased in hearts of Pdgfrb^MerCre mice at 7 and 14 days after TAC (Figure 4, A and B). Phosphorylation of ERK1/2, previously demonstrated to protect the heart in response to load stress (24), was significantly decreased at 14 but not 7 days after TAC in Pdgfrb^MerCre mice (Figure 4, A and C). A similar pattern was observed with respect to phosphorylation of JNK kinase (Figure 4A). In addition, levels of phosphorylation of the protein kinase Akt were significantly reduced in Pdgfrb^MerCre mice compared with Pdgfrb^fl/fl controls 14 days after exposure to TAC (Figure 4, D and E).

Figure 4

Inducible, cardiac-specific PDGFR-β–knockout mice exhibit impaired activation of Akt and MAPK pathways in the progression to heart failure. Representative Western blots from cardiac lysates of Pdgfrb^MerCre mice (inducible, cardiac-specific PDGFR-β–knockout mice) or Pdgfrb^fl/fl controls harvested prior to TAC or at indicated time points after TAC probed for (A) phospho-/total p38, phospho-/total ERK1/2, and phospho/total JNK or (D) phospho-/total Akt. (B, C, and E) Quantification by densitometry (n = 4 independent samples per group per time point) of ratios of (B) phospho-/total p38, (C) phospho-/total ERK1/2, or (E) phospho-/total Akt. (F) Number of apoptotic cells per 100,000 myocytes from hearts of Pdgfrb^MerCre mice or Pdgfrb^fl/fl controls assessed at 7 or 14 days after TAC. Data represent results from 2 spatially separated samples from 3 separate animals from each group at each time point. P values were determined by unpaired, 2-tailed Student’s t test.

We also observed diminished phosphorylation of both Akt and ERK1/2 in the hearts of Pdgfrb^Nkx-Cre mice compared with controls 14 days after TAC stress (Supplemental Figure 6). Since both Akt and ERK1/2 exert cardioprotective effects in part via inhibition of apoptosis, we sought to determine whether cardiomyocyte apoptosis after TAC was different in Pdgfrb^MerCre mice compared with Pdgfrb^fl/fl controls. Consistent with our biochemical findings, the number of apoptotic cardiomyocytes was increased in Pdgfrb^MerCre mice 7 days after TAC and continued to rise 14 days after TAC (Figure 4F). Overall, these findings suggest that PDGFR-β signaling in the heart may participate in modulating the activation of cardioprotective protein kinases in the progression of heart failure due to pressure overload.

Angiogenic gene expression profile correlates with defective microvascular function in inducible, cardiac-specific PDGFR-β–knockout mice upon exposure to load stress. The majority of the biochemical changes described in Figure 4 occurred after the initiation of the heart failure phenotype in Pdgfrb^MerCre mice upon exposure to pressure overload. To understand the initiating biological mechanism of heart failure in Pdgfrb^MerCre mice, we focused on identifying biologic changes between Pdgfrb^MerCre mice and Pdgfrb^fl/fl controls at time points preceding the development of overt heart failure using a broad approach, namely microarray gene expression studies. Of the genes that were differentially expressed (Supplemental Table 1), we focused on a group of genes that exhibited the pattern depicted in Figure 5A — genes expressed in the heart at similar levels in Pdgfrb^MerCre mice and Pdgfrb^fl/fl controls prior to TAC but increased in Pdgfrb^fl/fl controls compared with Pdgfrb^MerCre mice at early time points (3 and 7 days) after TAC. Functional annotation clustering of this subset of genes revealed an enrichment of genes that participate in multiple facets of angiogenesis (Figure 5B), including endothelial cell proliferation (e.g., Agtrl1; ref. 25), endothelial cell migration (Angpt1; ref. 26), extracellular matrix remodeling (e.g., Mmp2; ref. 27), and proangiogenic transcription factors (e.g., Elk3). We thus postulated that PDGFR-β signaling in the cardiomyocyte regulates cardiac angiogenesis in response to stress. This hypothesis correlates with reports indicating that the cardiomyocyte itself is a paracrine cell functioning to enhance blood vessel growth during conditions of increased substrate demand (28–30).

Figure 5

Angiogenic gene expression profile correlates with defective microvascular function in inducible, cardiac-specific PDGFR-β–knockout mice upon exposure to load stress. (A) Pattern of change of genes of interest over time prior to and after TAC (red, Pdgfrb^MerCre; blue, Pdgfrb^fl/fl). (B) Change of expression from baseline (ratio of Pdgfrb^MerCre to Pdgfrb^fl/fl) of genes associated with multiple aspects of angiogenesis (n = 4 samples in each group at each time point). (C) Representative ultrasound tracings of maximal coronary flow after hyperemic stimulus prior to TAC or 14 days after TAC in control Pdgfrb^fl/fl or Pdgfrb^MerCre mice. (D) Quantification of CFR in Pdgfrb^fl/fl, MerCreMer, or Pdgfrb^MerCre mice (n = 7 in each group at each time point). (E) Quantification of cardiac perfusion as assessed by myocardial contrast uptake (see Methods) from 0° (anterior wall of the left ventricle) to 90° (lateral wall of left ventricle) 14 days after TAC in Pdgfrb^MerCre or Pdgfrb^fl/fl control mice (n = 3 Pdgfrb^MerCre mice, n = 4 Pdgfrb^fl/fl control mice). P values were determined by ANOVA, and significant differences between Pdgfrb^MerCre and control (Pdgfrb^fl/fl and MerCreMer) mice were confirmed with Tukey’s test.

To test this hypothesis, we measured coronary flow reserve (CFR), which provides a robust assessment of coronary microvascular function in mice (31) and humans (32). Representative tracings (Figure 5C) of coronary flow in response to vasodilator stimulus (2.5% inhaled isoflurane; ref. 33) demonstrate reduced hyperemic coronary flow in Pdgfrb^MerCre mice at baseline and after TAC. Quantification of CFR demonstrated a significant reduction at baseline in Pdgfrb^MerCre mice compared with either MerCreMer or Pdgfrb^fl/fl controls (Figure 5D). By 7 days after TAC, Pdgfrb^MerCre mice had minimal augmentation of coronary flow in response to vasodilator stimulus (CFR = 1), suggestive of a severe coronary microvascular defect. To determine whether Pdgfrb^MerCre mice had a global decline in myocardial perfusion in response to TAC, we used a microbubble perfusion method, producing a contrast-enhanced ultrasound signal in the myocardium that is directly proportional to perfusion (34). Using this technique throughout all assessable portions of the heart (the anterior and lateral segments of the left ventricle), we observed a reduction in myocardial perfusion in Pdgfrb^MerCre mice compared with Pdgfrb^fl/fl controls (Figure 5E). Overall myocardial perfusion was reduced by 24.6% in inducible-mutant mice compared with controls 14 days after TAC (n = 3 Pdgfrb^MerCre mice, n = 4 Pdgfrb^fl/fl mice, P = 0.01).

Impaired angiogenesis and evidence of ischemic injury in the hearts of inducible, cardiac-specific PDGFR-β–knockout mice upon exposure to load stress. Recent work demonstrates that angiogenesis is a crucial adaptive mechanism accompanying cardiac hypertrophy and manifesting as an increased number of coronary microvessels per cardiomyocyte (29). To determine whether coronary angiogenesis in response to pressure overload was impaired in Pdgfrb^MerCre mice, we quantified coronary microvessels at baseline or after TAC exposure. The number of CD31 microvessels per myocyte at baseline was not different in the hearts of Pdgfrb^MerCre mice compared with Pdgfrb^fl/fl controls (Figure 6A). In contrast, while Pdgfrb^fl/fl and MerCreMer control mice had a rise in coronary microvessels per cardiomyocyte in response to TAC consistent with previous findings (29), this increase was significantly blunted in Pdgfrb^MerCre mice at 7 and 14 days after TAC. Representative photomicrographs from hearts 14 days after TAC demonstrate the reduction in microvascular density in hearts of Pdgfrb^MerCre mice compared with MerCreMer or Pdgfrb^fl/fl controls (Figure 6B).

Figure 6

Impaired angiogenesis and evidence of ischemic injury in the hearts of inducible, cardiac-specific PDGFR-β–knockout mice upon exposure to load stress. (A) Microvessel number per cardiomyocyte in Pdgfrb^fl/fl, MerCreMer, or Pdgfrb^MerCre mice at baseline (day 0) or at time points after TAC. (B) Representative photomicrographs of cardiac sections stained with an anti-CD31 antibody from Pdgfrb^fl/fl, MerCreMer, or Pdgfrb^MerCre hearts 14 days after TAC. Scale bars: 100 μm. (C) Low-power (original magnification, ×15) photomicrographs of representative Masson’s trichrome–stained cardiac sections from Pdgfrb^fl/fl or Pdgfrb^MerCre mice 14 days after TAC reveal diffuse, subendocardial fibrosis in hearts of Pdgfrb^MerCre exposed to load stress (upper panels). High-power photomicrographs of portion of sections in C indicated by boxed region (lower panels). Scale bars: 50 μm. (D) Representative photomicrographs of cardiac sections from Pdgfrb^MerCre or Pdgfrb^fl/fl control mice assessed for hypoxia (see Methods) 14 days after TAC. Scale bars: 50 μm. (E) Quantification of hypoxic area in hearts of Pdgfrb^MerCre or Pdgfrb^fl/fl control mice after TAC. Data represent mean of multiple fields from independent sections from 3 separate mice in each group at each time point.

Histologically, hearts from Pdgfrb^MerCre mice demonstrated a significant increase in fibrosis (Figure 6C) distributed throughout the subendocardium, the portion of the heart most susceptible to ischemic injury. To determine whether the reduction in angiogenesis resulted in functional myocardial ischemia, we used 2-nitroimidazole, an immunochemical marker for cardiac hypoxia (29). We observed a significant increase in myocardial hypoxia in Pdgfrb^MerCre mice 7 days after TAC (Figure 6E) and a more dramatic increase in hypoxia in the hearts of Pdgfrb^MerCre mice 14 days after TAC, visually demonstrated in Figure 6D. Biochemically, we observed increased levels of the transcription factor HIF-1α, whose expression increases in the ischemic heart (35), 7 days after TAC in Pdgfrb^MerCre mice compared with Pdgfrb^fl/fl controls (Supplemental Figure 7), along with increased levels of the HIF-1α–dependent factor VEGF-A. However, 14 days after TAC, both HIF-1α and VEGF-A levels were significantly reduced in the hearts of Pdgfrb^MerCre mice despite profound tissue hypoxia. Taken together, our findings demonstrate that loss of cardiomyocyte PDGFR-β signaling results in a functional and structural defect in coronary microvascular flow accompanied by evidence of global myocardial ischemia in response to pressure overload–induced stress.

Cardiomyocyte PDGFR-β is an essential regulator of the paracrine angiogenic capacity of cardiomyocytes. Our in vivo observations demonstrate that loss of cardiomyocyte PDGFR-β results in load-induced cardiac dysfunction associated with impairment in cardiac angiogenesis. Based upon our gene expression data (Figure 5, A and B), we hypothesized that PDGFR-β is a regulator of the paracrine angiogenic capacity, or angiogenic potential, of cardiomyocytes. To test this hypothesis, we isolated a highly purified population of neonatal cardiomyocytes from Pdgfrb^fl/fl mice (Figure 7A). In vitro infection of these cardiomyocytes with Ad-Cre, but not with Ad-GFP, resulted in deletion of the floxed allele, detectable by PCR of genomic DNA (Figure 7B). Cre-mediated deletion of the floxed PDGFR-β allele from isolated cardiomyocytes resulted in a marked decrease in mRNA encoding PDGFR-β (Figure 7C).

Figure 7

Cardiomyocyte PDGFR-β is an upstream regulator of the paracrine angiogenic capacity of cardiomyocytes. (A) FACS analysis of cardiomyocytes from Pdgfrb^fl/fl mice stained with anti–sarcomeric myosin antibody. (B) Genomic PCR of cardiomyocytes from Pdgfrb^fl/fl mice before infection (Ctrl) or after infection with Ad-GFP or Ad-Cre using primers for floxed allele (upper band) or deleted allele (lower band). (C) Quantitative RT-PCR for PDGFR-β expression (normalized to GAPDH) using RNA derived from cardiomyocytes from Pdgfrb^fl/fl mice before infection (Control) or after infection with Ad-GFP or Ad-Cre. (D) Relative tube-forming capacity of conditioned media from cardiomyocytes from Pdgfrb^fl/fl mice before infection (Control) or after infection with Ad-GFP or Ad-Cre with or without exogenous PDGF-BB. (E) Relative tube-forming capacity of conditioned media from cardiomyocytes from Pdgfrb^fl/fl mice before infection or after infection with Ad-GFP or Ad-Cre under normoxic or hypoxic conditions. (F) Relative tube-forming capacity of conditioned media from cardiomyocytes from wild-type mice before or after infection with Ad-GFP or Ad-Cre and subsequent exposure to hypoxia. (G) Relative endothelial cell proliferation in conditioned media from wild-type and Pdgfrb^fl/fl cardiomyocytes before or after infection with Ad-GFP or Ad-Cre and exposed to hypoxia. *P < 0.001. (H) Ratio of reduction in expression (quantitative RT-PCR, normalized to GAPDH) of proangiogenic genes in cardiomyocytes from Pdgfrb^fl/fl mice infected with Ad-Cre or Ad-GFP and exposed to hypoxia. *P < 0.05. Data were derived from 4 independent experiments.

Using this primary cardiomyocyte model, we first explored the ability of PDGF stimulation to enhance the ability of cardiomyocytes to support angiogenesis. In experiments in which cardiomyocytes from Pdgfrb^fl/fl mice were used, conditioned media from control, noninfected cardiomyocytes, Ad-GFP–infected cardiomyocytes, or Ad-Cre cardiomyocytes was harvested before and after treatment with PDGF-BB or vehicle control. Conditioned media from control or Ad-GFP cardiomyocytes demonstrated a marked increase in the capacity to promote endothelial tube formation (a surrogate assay to assess the angiogenic capacity of cardiomyocytes) after treatment with PDGF-BB (Figure 7D), an effect not directly attributable to PDGF-BB (see Media, white bars). In contrast, the angiogenic capacity of conditioned media from cardiomyocytes in which PDGFR-β was deleted by Ad-Cre infection was not enhanced by PDGF-BB treatment. These findings demonstrate that PDGF-BB potently enhances the paracrine angiogenic capacity of cardiomyocytes in a PDGFR-β–dependent manner.

To determine whether PDGFR-β is a required component of the angiogenic potential of cardiomyocytes under conditions of physiologic stress, we exposed control, Ad-GFP–infected, or Ad-Cre–infected neonatal cardiomyocytes from Pdgfrb^fl/fl mice to hypoxia, then harvested the conditioned media from these myocytes and tested its ability to support endothelial tube formation. Hypoxia significantly increased the capacity of conditioned media from control and Ad-GFP cardiomyocytes to support tube formation (Figure 7E). In contrast, we saw a reduction in the angiogenic capacity of conditioned media from Ad-Cre–infected, PDGFR-β–deleted cardiomyocytes upon exposure to hypoxic stress. To determine whether Ad-Cre infection had a direct toxic effect on the angiogenic capacity of cardiomyocytes, we infected control neonatal cardiomyocytes (not bearing the PDGFR-β floxed allele) with Ad-Cre or Ad-GFP and exposed these myocytes to hypoxia. As shown in Figure 7F, Ad-Cre infection had no effect on the paracrine ability of cardiomyocytes to support endothelial tube formation in response to hypoxia. As an independent measure of the PDGFR-β–dependent paracrine angiogenic capacity of cardiomyocytes, we found that conditioned media from Ad-Cre–infected cardiomyocytes from Pdgfrb^fl/fl mice exposed to hypoxia had an impairment in the ability to support in vitro endothelial cell proliferation compared with Ad-GFP or control cardiomyocytes (Figure 7G). Experiments using wild-type cardiomyocytes demonstrated that this impairment in promoting endothelial cell proliferation is not attributable to Ad-Cre infection (Figure 7G). Furthermore, we identified a series of factors known to enhance in vivo angiogenesis that demonstrated a trend toward reduced expression in Ad-Cre–infected, PDGFR-β–deficient cardiomyocytes compared with control, Ad-GFP–infected cardiomyocytes after both cell models were exposed to hypoxia in vitro (Figure 7H), including statistically significant reductions in expression of Cxcl1, Cxcl2, and Fgf1. Thus, our findings demonstrate that PDGFR-β is an essential regulator of the angiogenic capacity of cardiomyocytes in response to hypoxic stress, in part through regulation of a program of expression of angiogenic factors. These findings are consistent with and provide a direct mechanistic basis for the cardiac dysfunction and impairment of load stress–induced angiogenesis observed in cardiac-specific PDGFR-β–knockout mice.

We show here that PDGFR-β is an indispensable component of the cardiac response to pressure overload–induced stress. The robustness of our findings is reflected in the similarity of phenotype in 2 independent but overlapping knockout systems. Mechanistically, our findings suggest that PDGFR-β signaling is an important upstream regulator of the angiogenic potential of the cardiomyocyte in response to stress. The notion that PDGFR signaling regulates proangiogenic pathways has precedent in other systems. For example, PDGFR signaling in tumor-associated stromal cells exerts a proangiogenic effect in the tumor microenvironment (36). Furthermore, a recent report revealed that knockout of PDGFR-β in the epicardium inhibits normal development of epicardial coronary arteries (37).

PDGFR-β signaling in the cardiomyocyte may regulate angiogenesis in the heart in response to load-induced stress through several different mechanisms. Recent work has demonstrated that activation of the PI3K and MAPK pathways enhance the angiogenic capacity of cardiomyocytes via a HIF-1α/VEGF-A–dependent mechanism (30). Intriguingly, we observed an increase in levels of phosphorylated Akt and a decrease in levels of phosphorylated p38 in the hearts of Pdgfrb^MerCre mice prior to TAC compared with levels in hearts of Pdgfrb^fl/fl mice (Figure 4), although these changes did not achieve statistical significance when quantified by densitometry. Furthermore, we observed significantly decreased phosphorylation of Akt, p38, and ERK1/2 together with significantly decreased expression of HIF-1α and VEGF-A in Pdgfrb^MerCre mice 14 days after exposure to TAC stress. However, at earlier time points after TAC (day 7), expression of both HIF-1α and VEGF-A were increased in Pdgfrb^MerCre mice exposed to TAC compared with Pdgfrb^fl/fl control mice. By this time point, Pdgfrb^MerCre mice already exhibited cardiac dysfunction along with markedly reduced numbers of blood vessels per cardiomyocyte and functional hypoxia. These observations suggest that the PDGFR-β–dependent angiogenic capacity of cardiomyocytes is independent of HIF-1α/VEGF-A expression. Elucidation of the signaling pathways downstream of PDGFR-β that are essential regulators of the PDGFR-β–dependent angiogenic capacity of cardiomyocytes is a goal of our future studies, as is the determination of the precise identity of paracrine factors that mediate the PDGFR-β–dependent paracrine angiogenic capacity of cardiomyocytes. Importantly, cardiomyocyte PDGFR-β signaling may regulate aspects of the cardiac response to pressure overload other than angiogenesis (e.g., substrate utilization) that substantially contribute to heart failure in cardiac-specific PDGFR-β–knockout mice, which we will explore in future studies.

Our findings suggest that therapeutics that augment cardiomyocyte PDGFR signaling in the heart may be a novel strategy for the treatment of cardiac disease in which defective angiogenesis is a contributor to disease pathogenesis. Consistent with this hypothesis are prior studies that demonstrate that administration of PDGF-BB prevents adverse remodeling after experimental myocardial infarction (38, 39). More generally, our findings support the general notion that targeting cardiac angiogenesis through the PDGF/PDGFR system or via other means may be an effective approach to prevent the progression of multiple forms of heart failure (40), including “nonischemic” cardiomyopathies, in which functional and structural microvascular abnormalities are well described (41).

Our studies were inspired by observations of cardiac toxicity in cancer patients treated with PDGFR inhibitors. Our results provide a mechanistic rationale to aggressively treat hypertension and thus minimize vascular stress as a strategy to prevent heart failure due to anticancer PDGFR inhibitors. Based on our findings, we also speculate that patients with preexisting coronary disease at the macro- or microvascular level may be at increased risk for cardiomyopathy and heart failure associated with these drugs. Confirmation of such findings could provide the basis for a risk stratification tool in cancer patients being considered for treatment with PDGFR inhibitors and could be used to develop strategies to prevent adverse cardiac effects of PDGFR inhibitors.

It is important to note that targets other than PDGFR may be involved in cardiotoxicity due to anticancer PDGFR inhibitors. Both sunitinib and sorafenib can exert substantial off-target effects on other kinases (42), and these can be independent or additive to their effects on cardiomyocyte PDGFR signaling that result in clinical heart failure. Furthermore, cardiotoxicity due to imatinib has been demonstrated to involve Abl kinase (11, 43), while the role of PDGFR signaling in cardiotoxicity due to imatinib is unclear (44). Fundamentally, it is of critical importance to determine the contribution of the inhibitory effects on PDGFR to the clinical cardiac toxicity observed with RTK inhibitors whose targets include PDGFR. Such information will be useful to achieve the goal of designing highly efficacious, novel anticancer agents with minimal cardiotoxicity.

Finally, our studies highlight the opportunities for discovery that can be capitalized upon based upon cardiac toxicity of novel, targeted anticancer agents. Such toxicities may reveal biological pathways whose role in the regulation of cardiovascular physiology were previously unknown, and detailed exploration of such pathways may be useful for developing new, effective treatments for multiple forms of human heart failure.

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We thank T. Finkel (NIH), E.T.H. Yeh (University of Texas MD Anderson Cancer Center), and R. Schwartz (Texas A&M University) for helpful discussions. This work was supported in part by a Howard Hughes Medical Institute Physician-Scientist Early Career Award (to A.Y. Khakoo), a Physician-Scientist Award from the University of Texas MD Anderson Cancer Center (to A.Y. Khakoo), a Michael and Marriet Cyrus Scholar Award (to A.Y. Khakoo), a postdoctoral fellowship from the American Heart Association (to D. Ai), the NIH (HL076661, HL089792, AG17899 to M.L. Entman), and by the Wanamacker Foundation (to M.L. Entman).

Address correspondence to: Aarif Y. Khakoo, University of Texas MD Anderson Cancer Center, Department of Cardiology, 1515 West Holcombe, Unit 1451, Houston, Texas 77030, USA. Phone: (713) 563-3563; Fax: (713) 563-0462; E-mail: aykhakoo@mdanderson.org.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2010;120(2):472–484. doi:10.1172/JCI39434.

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