Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires Tgf-β

Mutations in sarcomere protein genes can cause hypertrophic cardiomyopathy (HCM), a disorder characterized by myocyte enlargement, fibrosis, and impaired ventricular relaxation. Here, we demonstrate that sarcomere protein gene mutations activate proliferative and profibrotic signals in non-myocyte cells to produce pathologic remodeling in HCM. Gene expression analyses of non-myocyte cells isolated from HCM mouse hearts showed increased levels of RNAs encoding cell-cycle proteins, Tgf-β, periostin, and other profibrotic proteins. Markedly increased BrdU labeling, Ki67 antigen expression, and periostin immunohistochemistry in the fibrotic regions of HCM hearts confirmed the transcriptional profiling data. Genetic ablation of periostin in HCM mice reduced but did not extinguish non-myocyte proliferation and fibrosis. In contrast, administration of Tgf-β–neutralizing antibodies abrogated non-myocyte proliferation and fibrosis. Chronic administration of the angiotensin II type 1 receptor antagonist losartan to mutation-positive, hypertrophy-negative (prehypertrophic) mice prevented the emergence of hypertrophy, non-myocyte proliferation, and fibrosis. Losartan treatment did not reverse pathologic remodeling of established HCM but did reduce non-myocyte proliferation. These data define non-myocyte activation of Tgf-β signaling as a pivotal mechanism for increased fibrosis in HCM and a potentially important factor contributing to diastolic dysfunction and heart failure. Preemptive pharmacologic inhibition of Tgf-β signals warrants study in human patients with sarcomere gene mutations.

Dominant mutations in sarcomere protein genes cause hypertrophic cardiomyopathy (HCM), a primary myocardial disorder characterized by myocyte enlargement, increased myocardial fibrosis, and impaired ventricular relaxation that predisposes patients to develop heart failure (1–5). The population incidence of sarcomere protein gene mutations is considerable (4–6), and recent epidemiologic studies document 7-fold increased risk of heart failure in the 40 million individuals estimated to carry these defects (4). Definition and inhibition of the signals that cause pathologic cardiac remodeling in response to sarcomere protein gene mutations may therefore have broad impact on human health.

Previous studies of HCM mutations in the myosin heavy chain gene showed that these enhance sarcomere biophysical properties (7–9) and alter cardiac relaxation (10) and calcium signaling in myocytes (11), events that are associated with widespread transcriptional changes (12). In addition to inducing changes within myocytes, the characteristic histopathology found in HCM hearts implies that sarcomere gene mutations also impact non-myocyte cells.

Myocardial fibrosis, the collagen-rich extracellular matrix that is presumed to derive from fibroblast-like cells within the heart is substantially increased in HCM (13–15). Both interstitial fibrosis, which surrounds individual myocytes, and focal fibrosis, the microscopic scars that replace dead myocytes, accrue in HCM. Increased myocardial fibrosis is maladaptive, and accumulation correlates with impaired cardiac relaxation (16) and increases the propensity for heart failure. Mechanisms by which myocyte-specific mutations in sarcomere protein genes increase myocardial fibrosis remain unknown.

To define profibrotic signals in HCM, we studied 2 mouse lines that carry different human mutations in the α-cardiac myosin heavy chain gene: Arg403Gln (referred to as α-MHC^403/+) (17) and Arg719Trp (referred to as α-MHC^719/+; Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI42028DS1). These mice exhibit protean manifestations of HCM, including hypertrophy and focal fibrosis in adulthood (17, 18). The hearts from young α-MHC^403/+ and α-MHC^719/+ mice, like human children with HCM mutations, have normal dimensions and histology throughout a period of clinical latency (denoted as prehypertrophic). Despite the absence of hypertrophy or HCM histopathology, our studies of RNA expression in LV tissues from prehypertrophic α-MHC^403/+ mouse hearts (12) demonstrated increased transcription of genes associated with fibrosis.

To characterize molecular signals that promote fibrosis in α-MHC^403/+ and α-MHC^719/+ mice, we used comprehensive transcriptional analyses of RNAs expressed in isolated myocytes and non-myocyte cells from prehypertrophic HCM hearts and from hearts with overt HCM. Guided by increased expression of RNAs in non-myocyte cells, we assessed cell proliferation in HCM hearts and targeted potential signaling molecules implicated in activating fibrosis in HCM. Using these data, we tested and report a pharmacologic strategy for extinguishing the emergence of pathologic remodeling in prehypertrophic HCM mice.

HCM models. We studied 2 mouse HCM models. We used the previously described α-MHC^403/+ mice (17, 18), and we produced what we believe to be a new model that carries the Arg719Trp mutation in the α-cardiac myosin heavy chain gene (α-MHC^719/+ mice), using homologous recombination technology (Supplemental Figure 1). Hearts from young (<20 weeks) α-MHC^719/+ mice, like those of juvenile α-MHC^403/+ mice, do not have LV hypertrophy or HCM histopathology and are designated prehypertrophic. By 35 weeks, α-MHC^719/+ mice exhibit both hypertrophy and HCM histopathology that is indistinguishable from that of adult α-MHC^403/+ mice.

Our prior studies (11) showed that cyclosporine A (CsA) administration to young α-MHC^403/+ mice accelerates the emergence of HCM (referred to as hypertrophic α-MHC^403/+csa mice). CsA-treated WT mice (WTcsa mice) develop neither hypertrophy nor histopathology. CsA-treated α-MHC^719/+ mice (referred to as hypertrophic α-MHC^719/+csa mice) showed accelerated development of increased LV wall thickness (LVWT), overt HCM histopathology, including increased myocardial fibrosis, and preserved cardiac function (Figure 1A and Table 1). The maximum LVWT was 1.8-fold increased in hypertrophic α-MHC^719/+csa mice compared with that of WTcsa mice (P = 3 × 10^–4). Masson trichrome–stained sections showed approximately 80-fold greater fibrosis in hearts (n = 3 per genotype) from hypertrophic α-MHC^719/+csa mice than WTcsa mice (4.4% ± 2.8% vs. 0.05% ± 0.07%, n ≥ 27 sections per genotype; P = 1.3 × 10^–11) and 40% more cardiac fibrosis than prehypertrophic α-MHC^719/+ mice (n = 7; 3.10% ± 3.1%; n ≥ 105 sections per genotype; P = 0.012; Figure 1A).

Figure 1

Hypertrophic α-MHC^719/+ mice have cardiac hypertrophy, increased myocardial fibrosis, and non-myocyte cell proliferation. (A) Masson trichrome–stained sections from prehypertrophic (Pre-) and hypertrophic (Hyp-) α-MHC^719/+csa hearts revealed increased fibrosis (blue) in comparison with WTcsa hearts. Scale bar: 1 mm. (B) Light microscopy shows increased BrdU incorporation in sections from α-MHC^719/+csa hearts (top panel, original magnification, ×50). Confocal immunofluorescence after WGA (green), DAPI (blue) stains, and BrdU antibodies revealed proliferating non-myocyte cells (magenta nuclei, arrowheads) in regions with increased fibrosis (bottom panel, original magnification, ×400). There was minor nonspecific antibody staining (*) outside the nuclei. In comparison with WTcsa heart sections, note that α-MHC^719/+csa hearts have disorganized myocardial architecture. Scale bar: 75 μm. (C) Confocal immunofluorescence shows expression of α-smooth muscle actin and fibroblast-specific protein 1 (Fsp1/S100a4), detected using a FITC-conjugated α-smooth muscle actin mouse antibody (green) and S100a4 antibody (red), respectively. Note that FITC-conjugated α-smooth muscle actin labeled only vascular beds in WTcsa and hypertrophic α-MHC^719/+csa hearts. Scale bar: 75 μm.

Table 1

Effects of Tgf-β inhibition or periostin ablation on cardiac morphology and function in HCM mice

RNA profiles of isolated cardiac myocytes and non-myocyte cells. To define signaling pathways that promote fibrosis in response to a sarcomere gene mutation, RNA expression was assessed by deep sequence analysis of gene expression (DSAGE; see Methods) in myocytes and non-myocyte cells isolated from hearts of 9- to 12-week-old WT, prehypertrophic α-MHC^403/+, WTcsa, and hypertrophic α-MHC^403/+csa mice. Of 15,579 distinct RNAs found in non-myocyte cells, 7,578 RNAs were expressed at higher levels than in myocytes. Among RNAs that were significantly enriched in non-myocyte cells (P < 0.001), 1,317 changed with hypertrophic remodeling (Supplemental Table 1); expression decreased in 272 RNAs and increased in 1,045 RNAs. Non-myocyte RNAs that were significantly increased (P < 0.0001) in hypertrophic α-MHC^403/+csa mice compared with WTcsa mice (Supplemental Tables 1 and 2) included transcripts encoding extracellular matrix proteins that have been previously identified in fibroblasts from other tissues, such as periostin (Postn), Tgfb, connective tissue growth factor (Ctgf), collagens (19), vimentin, chemokines (20), and metalloproteinases (21).

Using the DAVID (version 6.7) (22, 23) functional annotation tool, we identified 18 Gene Ontology (GO) terms (Supplemental Table 3) that were significantly overrepresented (P < 0.05 after Bonferroni correction) among differentially expressed genes in non-myocyte cells (Supplemental Table 1). This unbiased analysis corroborated our interpretation of DSAGE data, in that GO terms related to extracellular matrix, cell-cycle control, and cell proliferation were enriched in non-myocyte cell RNAs (Supplemental Table 3). Notably, 50 RNAs encoding proteins involved in cell-cycle control had significantly altered levels (P = 0.01 after Bonferroni correction; see bolded RNAs in Supplemental Tables 1 and 3). Based upon these transcriptional profiles, we concluded that non-myocyte cells in hypertrophic α-MHC^403/+csa hearts were activated.

Proliferation and characterization of activated non-myocyte cells. To obtain in vivo confirmation of activated pathways in non-myocyte cells during hypertrophic remodeling, we assessed cell proliferation in α-MHC^403/+csa and α-MHC^719/+csa mice, using incorporation of BrdU to detect newly synthesized DNA and nuclear expression of Ki67 to detect cell-cycle activity (20, 24, 25). BrdU was injected 3 days prior to sacrifice of hypertrophic mice and sections were stained with BrdU and Ki67 antibodies (Figure 1B; see Methods). Results from these independent methodologies were concordant, but as expected, absolute numbers of Ki67 antigen-labeled cells were consistently lower than those of BrdU-labeled cells. As in other studies (26, 27), we found no evidence for myocyte proliferation; BrdU and Ki67 antibodies labeled only non-myocyte cells.

There were 4-fold more BrdU-labeled non-myocyte cells from hypertrophic α-MHC^719/+csa LV sections than from prehypertrophic or WTcsa LV sections (P = 3 × 10^–12). In regions with normal histologic architecture (e.g., absence of focal fibrosis or expansion of the interstitium) from WTcsa and prehypertrophic α-MHC^719/+ mice, amounts of BrdU-labeled non-myocyte cells were similar (0.10%–0.14%, >155,000 cells per genotype), but these were 3.3-fold increased in hypertrophic α-MHC^719/+csa mice (0.42% ± 0.33%, >97,000 cells; P = 3 × 10^–8). In LV sections with focal fibrosis or overt interstitial expansion (identified by wheat germ agglutinin [WGA]) from hypertrophic α-MHC^719/+csa mice, BrdU labeled 1.8% ± 1.6% of non-myocyte cells (>110,000 cells), indicative of 4-fold greater regional proliferation of cells (Figure 1B).

We confirmed that non-myocyte proliferation in hypertrophic hearts from α-MHC^719/+ mice (age >35 weeks) that did not receive CsA. Ki67 antigen labeled 1.1% ± 0.56% of non-myocyte cells in regions with focal fibrosis and expanded interstitium (identified by WGA) compared with 0.61% ± 0.34% of non-myocyte cells in regions where the myocardial architecture was preserved (n = 3 mice, >19,000 cells per group; P = 0.001).

Proteins involved in the fibrotic response to sarcomere protein gene mutations. Expression of α-smooth muscle actin and fibroblast-specific protein (Fsp1, also known as S100a4), encoded by Acta2 and S100a4, respectively, is associated with some fibroblast populations (28–30). We used antibodies to these proteins (Figure 1C) to study hearts from hypertrophic α-MHC^719/+csa and WTcsa mice. The α-smooth muscle actin antibody stained perivascular smooth muscle cells but not the non-myocyte cells residing throughout the LV. In contrast, Fsp1 antibody labeled few non-myocyte cells in WTcsa hearts but substantial numbers of non-myocyte cells in hypertrophic α-MHC^719/+csa LV sections, particularly in fibrotic regions in which 28% of proliferating cells were Fsp1 positive (Supplemental Figure 2).

Multiple RNAs that encode molecules involved in extracellular matrix biology were enriched in non-myocyte cells isolated from prehypertrophic and hypertrophic α-MHC^403/+csa hearts (Supplemental Table 1). Among the genes that had the most significantly increased expression (≥7 fold) in non-myocyte cells from hypertrophic α-MHC^403/+csa hearts, we identified several Tgf-β–responsive genes (Supplemental Table 2, indicated in bold), including Postn (periostin). We confirmed increased periostin protein levels by Western blots of LV extracts. Periostin levels in extracts from prehypertrophic α-MHC^719/+csa, WT, and WTcsa mice were low (Figure 2A), but levels were markedly higher among age-matched hypertrophic α-MHC^719/+csa mice. The amount of increase in periostin protein was variable among hypertrophic α-MHC^719/+csa LV extracts, a finding that is consistent with our earlier evidence that fibrotic load differs among identical HCM mice (18).

Figure 2

Increased periostin protein in hypertrophic α-MHC^403/+ and α-MHC^719/+ mice. (A) Western blots detected periostin protein (arrow) in LV extracts from WT, prehypertrophic α-MHC^719/+, WTcsa, and hypertrophic α-MHC^719/+csa mice but not in extracts from Postn^–/– mice (data not shown). Gapdh served as a control for loading. Periostin levels were variable in hypertrophic α-MHC^719/+csa mice, but densitometry indicated significant increases (P = 0.035) above levels in WTcsa mice. (B) Histopathological comparison of fibrotic regions in hearts from Postn^–/–csa, hypertrophic α-MHC^403/+csa, and Postn^–/–α-MHC^403/+csa mice. Scale bar: 1 mm. (C) Immunofluorescent images of LV sections stained for Ki67 antigen (magenta) and DAPI (blue) indicated proliferating cells in fibrotic areas (identified by WGA, green). Scale bar: 75 μm. Numbers of Ki67 antigen-positive nuclei in fibrotic regions of Postn^–/–α-MHC^403/+csa mice were significantly less than those in α-MHC^403/+csa mice.

To determine whether periostin was essential for pathologic remodeling in HCM, we used a genetic strategy. α-MHC^403/+ mice were crossed with periostin-null mice (Postn^–/– mice ) (31), hypertrophy was induced with CsA, and compound mutant mice were studied by echocardiography and histopathology. WTcsa and Postn^–/–csa mouse (n = 5 per genotype) hearts were indistinguishable, with comparable maximal LVWT, normal cardiac histology, and very little fibrosis (Figure 2B). Maximal LVWT was modestly but not significantly reduced in hypertrophic Postn^–/–α-MHC^403/+csa mice (0.97 ± 0.37 mm) compared with that of hypertrophic α-MHC^403/+csa mice (1.21 ± 0.27 mm; Table 1). Masson trichrome–stained LV sections (Figure 2B) showed less myocardial fibrosis in hypertrophic Postn^–/–α-MHC^403/+csa hearts (2.06% ± 2.0%, n = 5) than hypertrophic α-MHC^403/+csa hearts (3.12% ± 3.38%, n = 6; P = 0.03; n >57 sections per genotype).

To determine whether periostin ablation altered non-myocyte proliferation in HCM hearts, we immunostained heart sections for nuclear Ki67 antigen (Figure 2C). Within fibrotic foci, periostin ablation reduced the number of Ki67-positive non-myocyte cells by 2.4-fold (hypertrophic Postn^–/–α-MHC^403/+csa mice, 0.55% ± 0.5%; n > 125,000 cells vs. hypertrophic α-MHC^403/+csa mice, 1.33% ± 0.6%; n > 150,000 cells; P = 6 × 10^–14). However, the fraction of Ki67-positive nuclei in the LV of Postn^–/–α-MHC^403/+csa mice (0.33% ± 0.27%; n > 235,000 cells) remained significantly elevated over that of control specimens (WTcsa, 0.05% ± 0.09%; n > 96,000 cells; P = 3.4 × 10^–43; Postn^–/–csa, 0.06% ± 0.11%; n > 98,000 cells; P = 3 × 10^–45). Based upon cardiac imaging, histopathology, and non-myocyte proliferation in periostin-null hypertrophic mice, we concluded that periostin contributed to, but was not essential for, pathologic remodeling in HCM.

To address the roles of Tgf-β signaling in HCM, we administered Tgf-β–neutralizing antibodies (Tgf-β NAbs) or rabbit IgG prior to and throughout CsA treatment of α-MHC^719/+ mice. The maximum LVWT of Tgf-β NAb–treated α-MHC^719/+csa mice (0.77 ± 0.1 mm) was significantly less than that of IgG-treated hypertrophic α-MHC^719/+csa mice (1.19 ± 0.2 mm; P = 0.009; Table 1).

There was also significantly less cardiac fibrosis in histological sections from Tgf-β NAb–treated mice than in those from IgG-treated hypertrophic α-MHC^719/+csa mice (n = 4 per treatment group; Figure 3A). Fibrosis areas encompassed 0.38% ± 0.4% of LV sections in Tgf-β NAb–treated α-MHC^719/+csa hearts compared with 1.77% ± 1.8% in IgG-treated hypertrophic α-MHC^719/+csa hearts (n = 60 sections per treatment group; P = 4 × 10^–8). Immunohistochemical staining showed that mice treated with Tgf-β NAb but not rabbit IgG, had reduced periostin expression in hypertrophic LV sections (Figure 3B). Moreover, Tgf-β NAb–treated α-MHC^719/+csa mice had significantly less (P = 1.7 × 10^–10) non-myocyte proliferation (BrdU labeling assessed in >120,000 cells per treatment group; Figure 3C) in regions with fibrosis (0.66% ± 0.06%) and regions with preserved myocardial architecture (0.13% ± 0.01%) as compared with IgG-treated mice (fibrosis, 1.87% ± 0.17%; preserved architecture, 0.62% ± 0.05%). Taken together, Tgf-β NAb attenuated hypertrophy, abrogated periostin expression, and reduced non-myocyte proliferation in HCM mice.

Figure 3

Tgf-β NAb attenuated cardiac fibrosis, reduced periostin expression, and diminished non-myocyte proliferation in hypertrophic α-MHC^719/+csa mice. (A) Histopathologic sections of Tgf-β NAb–treated and rabbit (Rb) IgG-treated α-MHC^719/+csa mice stained with Masson trichrome (blue) demonstrated less fibrosis in Tgf-β NAb–treated mice. Scale bar: 1 mm. (B) Immunofluorescent images of LV sections stained for periostin (green) and WGA (red) showed reduced periostin expression in Tgf-β NAb–treated α-MHC^719/+ mice and smaller areas of fibrosis compared with rabbit-IgG treated mice. Scale bar: 0.5 mm. (C) Confocal immunofluorescent images of LV sections from BrdU-treated mice, analyzed using BrdU antibodies (magenta), WGA (green), and DAPI (blue), showed that Tgf-β NAb treatment reduced the numbers of proliferating cells in fibrotic areas. Scale bar: 75 μm.

To identify downstream mediators of Tgf-β signaling in HCM, we assessed the location and phosphorylation of Smad2 (pSmad2) in LV sections from mice (n = 3 per genotype) (Supplemental Methods). Nuclear pSmad2 was found both in non-myocyte cells and myocytes in α-MHC^719/+csa LV sections. Immunohistochemistry (Supplemental Figure 3) detected approximately 50% (P = 9.7 × 10^–174) more non-myocyte nuclei stained with anti-phosphorylated Smad2 (pSmad2-Ser465/467) in regions with fibrosis (73.2% ± 14.6%, >7,500 cells) as compared with regions with normal myocardial architecture (48.4% ± 15.9%, >6,400 cells). Based on these data, we deduced that HCM mutations activated the canonical Tgf-β signaling pathway in non-myocyte cells.

We extended these studies by harnessing a pharmacologic approach to inhibiting Tgf-β signals. Recent data demonstrate that angiotensin II promotes cardiac fibrosis in part by activating Tgf-β signals (32). Moreover, the angiotensin II type I receptor antagonist losartan has had salutary effects on animal models of human diseases, including CsA-induced nephropathy (33), Marfan syndrome (34), skeletal myopathies (35), and transgenic overexpression of a cardiac troponin T mutation (36). To assess the effects of losartan in HCM, we treated prehypertrophic α-MHC^719/+ mice for 2 weeks prior to and during CsA induction of HCM. There was no significant difference in the conscious blood pressures of CsA-treated α-MHC^719/+ mice, with or without losartan (0.44 mg/d; P = NS; data not shown). However, the maximum LVWT (Table 1) of losartan-treated α-MHC^719/+csa mice (0.64 ± 0.03 mm) was comparable with that of WTcsa mice (0.73 ± 0.13 mm; P = NS) and significantly less (P = 7 × 10^–4) than that of untreated hypertrophic α-MHC^719/+csa mice (1.33 ± 0.17 mm). Compared with untreated hypertrophic α-MHC^719/+csa mice (n = 4), losartan-treated α-MHC^719/+csa mice (n = 3) also had 2.9-fold less fibrosis in LV sections (5.27% ± 0.17% vs. 1.80% ± 0.19%, n ≥ 45 sections per group; P = 1 × 10^–24; Figure 4A). Consistent with the results of Tgf-β NAb studies, periostin expression was substantially reduced in losartan-treated α-MHC^719/+csa mice (data not shown). Losartan also inhibited non-myocyte proliferation (Figure 4B). Fewer Ki67-positive non-myocyte cells were observed in losartan-treated mice versus untreated α-MHC^719/+csa mice, both in fibrotic regions (treated, 0.14% ± 0.12%, >69,000 cells; untreated, 0.86% ± 0.6%, >120,000 cells; P = 5 × 10^–12) and in regions with preserved myocardial architecture (treated, 0.04% ± 0.06%; >79,000 cells; untreated, 0.19% ± 0.25%; >79,000 cells; P = 2 × 10^–4).

Figure 4

Losartan attenuated cardiac fibrosis, reduced periostin expression, and diminished non-myocyte proliferation in hypertrophic α-MHC^719/+csa mice. (A) Histopathologic sections stained with Masson trichrome stain (blue) revealed minimal fibrosis in losartan-treated α-MHC^719/+csa compared with untreated α-MHC^719/+ csa mice. Scale bar: 1 mm. (B) Confocal immunofluorescence images of LV sections stained for Ki67 antigen (magenta), WGA (green), and DAPI (blue) showed that losartan reduced non-myocyte proliferation. Scale bar: 60.6 μm. (C) Cardiac sections stained with Masson trichrome after chronic (30 weeks) losartan treatment in α-MHC^719/+ mice showed markedly reduced amounts of myocardial fibrosis compared with untreated age-matched α-MHC^719/+ mice. Note that these 35-week-old α-MHC^719/+ mice did not receive CsA. Scale bar: 1 mm.

To determine whether losartan could attenuate the insidious pathologic remodeling that naturally emerges in α-MHC^719/+ mice after 30 weeks (without CsA), we chronically treated prehypertrophic α-MHC^719/+ mice with losartan, beginning at 5 weeks of age. Untreated α-MHC^719/+ mice developed hypertrophy by 30 weeks of age (maximal LVWT, 1.13 ± 0.02 mm; Table 2). In contrast, long-term losartan-treated α-MHC^719/+ mice had cardiac dimensions (maximal LVWT, 0.90 ± 0.02 mm; P = 2 × 10^–10; Table 2) that were indistinguishable from those of WT mice (maximal LVWT, 0.89 ± 0.02 mm). Histopathological analysis (Figure 4C) showed little fibrosis in chronic losartan-treated α-MHC^719/+ mice (0.34% ± 0.04%, n = 7 mice, 105 sections) in comparison with untreated 35-week-old α-MHC^719/+ mice (1.34% ± 0.52%, n = 10 mice, 150 sections).

Table 2

Effect of chronic losartan on the emergence of HCM in α-MHC^719/+ mice

We then asked whether losartan could reverse established hypertrophy and fibrosis in hypertrophic α-MHC^719/+csa mice. Nine- to fourteen-week-old hypertrophic α-MHC^719/+csa mice (n = 4, CsA treatment for 3–4 weeks) were treated with a high dosage of losartan (2.4 mg/d; see Methods) for 4 weeks and compared with untreated age-matched α-MHC^719/+csa (n = 4) and WTcsa (n = 5) mice. The area of fibrosis in LV sections from losartan-treated mice did not differ from that of hypertrophic α-MHC^719/+csa hearts (treated, 10.99% ± 4.08%; untreated, 10.18% ± 5.59%; n ≥ 60 sections per group; P = NS; Figure 5A and data not shown). However, the percentage of BrdU-positive nuclei (Figure 5B) in fibrotic regions from untreated mice (3.27% ± 3.65%; n > 100,000 cells) was 8-fold greater than that in fibrotic regions of losartan-treated hearts (0.41% ± 0.24%; n > 100,000 cells; P = 1.4 × 10^–7). Losartan treatment reduced the percentage of BrdU-positive nuclei in hypertrophic α-MHC^719/+csa hearts (0.28% ± 0.16%; n > 200,000 cells) to levels found in WTcsa hearts (0.27% ± 0.17%; n > 130,000 cells; P = NS).

Figure 5

Losartan did not reverse fibrotic remodeling in established HCM but diminished non-myocyte proliferation. (A) Cardiac sections from hypertrophic α-MHC^719/+csa mice, treated or not treated with losartan, stained with Masson trichrome showed comparable amounts of myocardial fibrosis. Scale bar: 1 mm. (B) Non-myocyte proliferation, assessed by BrdU incorporation, was significantly reduced by losartan treatment compared with that of untreated mice in regions of fibrosis (WGA, green; DAPI, blue). Arrowheads indicate BrdU-positive nuclei. Scale bar: 75 μm.

We demonstrate that myocyte expression of sarcomere protein mutations alters gene transcription in non-myocyte cells, inducing proliferation and expression of profibrotic molecules that produce pathologic remodeling in HCM. Tgf-β signals are essential to activating non-myocyte cells: inhibition of these signals, directly by Tgf-β NAbs or indirectly by the angiotensin II type 1 receptor antagonist losartan, ameliorated hypertrophy and HCM histopathology in mice. These data indicate a potential therapeutic strategy for patients with sarcomere protein gene mutations.

Amounts of myocardial fibrosis in human HCM hearts correlate with the degree of hypertrophy (37), diminished ventricular performance (38), diastolic dysfunction (16), and energy demands (39) — factors that contribute to heart failure and may increase arrhythmic risk (40, 41). As such, the benefit from inhibiting fibrosis in HCM could be substantial.

Fibrosis accrues in HCM hearts due to premature death of mutant myocytes and expansion of the interstitial matrix. Compromised coronary flow due to hypertrophy, microvascular dysfunction (42), increased oxidative stress (43), and increased metabolic demands imposed by abnormal biophysical properties of mutant sarcomeres (7, 8) are factors that contribute to premature myocyte death and the emergence of focal fibrosis in HCM. Far less is known about mechanisms that expand the extracellular matrix in HCM hearts. We demonstrated 4- and 3-fold increased non-myocyte proliferation in fibrotic regions (with focal scarring and/or expanded interstitium) and in areas of preserved myocardial architecture, respectively, in overt HCM compared with prehypertrophic α-MHC^719/+ hearts. Although we used a pharmacologic strategy to accelerate the emergence of HCM, the very low proliferation rates of non-myocyte cells in CsA-treated WT mice excluded the likelihood that increased cell division was a drug-induced, mutation-independent response. Instead, we suggest that there is insidious proliferation of non-myocyte cells in untreated HCM mice and human patients. Moreover, transcriptional profiling of non-myocyte cells indicated that proliferation was coupled to increased expression of profibrotic molecules (including collagens, periostin, elastin; Supplemental Table 2), a sequence that could readily expand the extracellular matrix in HCM hearts (Figure 6) and contribute to the progressive diminution of diastolic function observed in human HCM (44).

Figure 6

A model for increasing fibrosis and diastolic dysfunction from HCM sarcomere gene mutations. Mutant myocytes have increased biophysical properties (8) and abnormal Ca²⁺ homeostasis (11), factors that trigger mechanical and/or biochemical signals that activate gene transcription, including increased Tgf-β expression. Whether by paracrine and/or autocrine signaling, Tgf-β stimulates non-myocyte proliferation and expression of profibrotic molecules. Activated non-myocyte cells secrete profibrotic molecules that expand the interstitium, increase stresses imposed on mutant myocytes, and promote myocyte death with resultant focal scarring. Tgf-β–mediated increased interstitial and focal fibrosis contributes to diastolic dysfunction in HCM hearts. Preemptive antagonism of Tgf-β signaling by a neutralizing antibody (NAb) or losartan reduces non-myocyte proliferation and profibrotic gene expression, thereby limiting cardiac fibrosis.

Our studies did not address whether resident or newly recruited cells become activated in HCM. Tgf-β signals can stimulate cardiac microvascular endothelial cells to undergo endothelial-to-mesenchymal transformation, with migration into the myocardium and expression of genes such as Acta2 (14) and Fsp1 (45). Fsp1 but not α-smooth muscle actin was found in non-myocyte cells from hypertrophic hearts (Figure 1C). Activated non-myocyte cells may also be derived from circulating cells (14, 46–48). Alternatively, the enhanced biomechanical forces resulting from sarcomere protein mutations (7, 8) could also provide a local mechanism for activating resident non-myocyte cells. Lineage studies are underway to assess the source of activated non-myocyte cells in HCM hearts.

We identified periostin and Tgf-β as potentially critical molecules to non-myocyte activation. Periostin has been previously proposed to enable myocyte reentry into the cell cycle (49); however, like others (26), we found no evidence for myocyte proliferation, despite periostin expression in HCM hearts. Periostin also promotes differentiation of circulating cells into cardiac fibroblasts (50), stimulates production of collagen and extracellular matrix production (51), and promotes myocardial healing and scar formation after injury (31, 52). These functions might be presumed to be beneficial in HCM, given premature death of mutant myocytes. However, genetic ablation of periostin from HCM mice had no deleterious consequences on survival or cardiac dimensions, despite significantly reducing non-myocyte proliferation in fibrotic foci. Moreover proliferation indices in Postn^–/–α-MHC^403/+ HCM mice were still significantly higher than those in Postn^–/– mice, suggesting the involvement of other molecules in non-myocyte proliferation and activation in HCM hearts. This conclusion is supported by studies of Postn^–/– mice subjected to pressure overload, which still developed fibrosis and hypertrophy, albeit more slowly than pressure-overloaded WT mice (31).

In contrast to periostin, we identified Tgf-β signals as critical for pathologic remodeling in HCM. Transcript levels of Tgf-β were increased in both prehypertrophic and hypertrophic hearts. Treatment of HCM mice with Tgf-β NAb extinguished non-myocyte proliferation (Tgf-β NAb–treated, 0.13% ± 0.01%; WT, 0.10%–0.14%), markedly diminished expression of the pro-fibroblast differentiation molecule periostin (Figure 3), and attenuated the emergence LV hypertrophy. Notably, the decrease in non-myocyte proliferation or periostin expression in Tgf-β NAb–treated HCM hearts had no adverse impact on LV morphology (e.g., LV wall thinning or ventricular dilatation) or on systolic function (Table 1).

The source of increased Tgf-β in HCM hearts may be myocytes or non-myocyte cells. Prehypertrophic myocytes had lower levels of Tgf-β transcripts than non-myocyte cells (data not shown) and may reflect some contamination of non-myocyte cells in isolated myocyte preparations (estimated as <10% by immunohistochemical studies). Alternatively, modest expression of myocyte Tgf-β might occur in response to enhanced biophysical properties of mutant sarcomeres (8), similar to increased Tgf-β expression that occurs in cultured WT myocytes subjected to mechanical stretching (53). Biophysical forces of HCM hearts could similarly account for increased Tgf-β expression in non-myocyte cells, as has been observed when non-myocyte cardiac cells are stretched ex vivo (54, 55).

In the lung and skin, fibrotic remodeling occurs from activation of latent Tgf-β in the extracellular matrix, rather than from increased transcription (56). Mechanical forces can activate latent Tgf-β, in part through αv-containing integrins (57), which respond to local cell-environment interactions. While our studies did not directly address this mechanism for increased Tgf-β signaling in HCM, we note that transcriptional profiling (Supplemental Table 1) showed enriched expression of αv-containing integrins in non-myocyte cells of the heart and increased expression of these during hypertrophic remodeling. As such, we suspect that both increased Tgf-β transcription and activation contribute to pathologic remodeling in HCM.

Tgf-β signaling activates a canonical, Smad-dependent pathway as well as Smad-independent pathways (58). We found heterogeneous nuclear accumulation of pSmad2 in HCM (Supplemental Figure 3) occurring in regions with focal fibrosis and expanded interstitial matrix to a greater extent than in regions with preserved myocardial architecture. Similar activation of the canonical Tgf-β signaling has been reported in Marfan syndrome (34), inherited muscular dystrophy (35), and autosomal recessive cutis laxa type I (59).

The angiotensin II type 1 receptor inhibitor losartan can limit Tgf-β activation (60) and reduce circulating Tgf-β (61). In our studies, losartan like Tgf-β NAb prevented activation of profibrotic pathways in CsA-accelerated HCM (Figure 4A) and also prevented the spontaneous hypertrophic remodeling that gradually emerges with age in untreated mutant mice (Figure 4C). Although losartan failed to reverse hypertrophy or fibrosis in established HCM (Figure 5), it did reduce numbers of proliferating non-myocyte cells. Ongoing studies will determine whether longer treatment is more beneficial.

Collectively, these data define a mechanism by which sarcomere mutations increase myocardial fibrosis and hypertrophy in HCM through increased expression of Tgf-β and activation of the canonical signaling pathway, leading to proliferation of non-myocyte cells and expression of profibrotic molecules (Figure 6). We suggest that this mechanism implicates Tgf-β signaling in progressive diastolic dysfunction, the fundamental hemodynamic abnormality observed in HCM that accounts for patient symptoms and outcomes. Whether suppression of Tgf-β activation in HCM hearts will attenuate relaxation abnormalities remains an important question.

While these mouse models do not perfectly mimic human disease, our results indicate a new strategy for limiting HCM pathophysiology, early inhibition of Tgf-β signaling. With the availability of robust platforms for detecting HCM mutations, identification of individuals with sarcomere protein gene mutations without overt disease and preemptive reduction of Tgf-β activation is feasible and may improve outcomes in HCM. We suggest that study of this strategy is warranted.

View Supplemental data

This work was supported by grants from Deutsche Forschungsgemeinschaft and Deutsche Stiftung für Herzforschung (to J.P. Schmitt), HHMI (to C.E. Seidman and J.D. Molkentin), the Leducq Fondation (to R. Markwald, C.E. Seidman, and J.G. Seidman), NHLBI (to R. Markwald, C.E. Seidman, and J.G. Seidman), and NIH (to J.G. Seidman). R. Markwald, R. Norris, and S. Hoffman were supported by the following grants: NIH-NHLBI HL33756, NIH-NCRR COBRE P20RR016434, ARRA Administrative Supplement for Translational Research, NIH-NCRR COBRE P20RR021949, the Foundation Leducq (Paris, France) Transatlantic Mitral Network of Excellence Grant 07CVD04, and the National Science Foundation: EPS-0902795. Their work was conducted in a facility constructed with support from NIH C06 RR018823 from the Extramural Research Facilities Program of the National Center for Research Resources.

Address correspondence to: J.G. Seidman, Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 256, Boston, Massachusetts 02115, USA. Phone: 617.432.7871; Fax: 617.432.7832; E-mail: seidman@genetics.med.harvard.edu.

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

Reference information: J Clin Invest. 2010;120(10):3520–3529. doi:10.1172/JCI42028.

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