Heart repair by reprogramming non-myocytes with cardiac transcription factors

Abstract

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

Figure 1. Reprogramming fibroblasts toward a cardiac phenotype in vitro by GHMT

a. and b. Immunofluorescent staining for cardiac markers, α-MHC-GFP and α-actinin. Adult CFs isolated from α-MHC-GFP reporter mice were transduced by retroviruses carrying GHMT or empty vector alone. Immunocytochemistry was performed at (a) 14 days or (b) 30 days following transduction. Three types of iCLMs were categorized based on cardiac gene expression and morphology. Type A iCLMs only express α-MHC-GFP (top panels). Type B iCLMs express both α-MHC-GFP and α-actinin, but do not display sarcomeric structures (middle panels). Type C iCLMs express both α-MHC-GFP and α-actinin, and display sarcomeric structures (bottom panels). Sarcomeres were more organized at day 30 than at day 14 post-transduction. White boxes are enlarged in insets. Scale bar, 20 μm. c. Quantification of Type A, B, and C iCLMs in adult CFs and TTFs 14 days after transduction with GHMT. Ten fields were randomly chosen from each experiment. Three independent experiments were performed. Data are presented as mean ± std. d. Representative calcium transient traces from the indicated cell types depicted as Fura-2 ratios (340/380 nm). iCLMs were derived from adult CFs and displayed a pattern of calcium transients most similar to neonatal ventricular cardiomyocytes. Adult CFs do not display calcium transients.

Figure 2. Reprogramming non-cardiomyocytes toward a cardiac fate in vivo by GHMT

a. Heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. GFP-infected myocardium showed only β-gal⁺ non-cardiomyocytes, GHMT-infected myocardium showed extensive β-gal⁺ non-cardiomyocytes and cardiomyocytes. Black boxes in top panels are enlarged in lower panels. Scale bar, 40 μm. b. cTnT immunostaining and X-gal staining of heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. The same cell is marked with the same number. Several β-gal⁺ cells in GHMT-infected injured hearts expressed cTnT and displayed organized sarcomere structure. White boxes are enlarged in insets. Sections of injured hearts were taken at the border zone. Scale bar, 40 μm. c. Quantification of β-gal⁺ cardiomyocytes in border zones and infarct zones from GFP-infected (168 sections, n=3) and GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice (20 sections, n=2) post-MI. Sections were taken at 4 levels with an interval of 250 μm below LAD ligation site. Data are presented as mean ± std. d. Staining of border zone from GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice post-MI by X-gal (blue), anti-cTnT (red) and anti-Cx43 (green). Gap junctions (green) were observed between β-gal⁺ and β-gal⁻cardiomyocytes (A and B) and between β-gal⁺ cardiomyocytes (C and D). The same cell is marked with the same letter. Scale bar, 20 um. e. Contractility and Ca²⁺ transients of β-gal⁺- and β-gal⁻ -cardiomyocytes. β-gal⁺-cardiomyocytes (iCLMs) were labeled with a fluorogenic β-gal substrate C₁₂FDG (green). Traces of sarcomere shortening were recorded from field-stimulatedβ-gal⁻-cardiomyocytes (n=15) and iCLMs (n=7) with clear striated morphology. 71.4% of iCLMs displayed a similar pattern of contractility and Ca²⁺ transients to β-gal⁻-cardiomyocytes. 28.6% of iCLMs demonstrated immature contractility.

Figure 3. Lineage tracing of GHMT-induced iCLMs in vivo

a. Border zone of heart sections isolated from tamoxifen treated Tcf21^iCre/Rosa26R^tdT mice that were subjected to LAD ligation followed by injection of GFP or GHMT retroviruses. Three weeks post-MI, hearts were fixed, sectioned and stained for cTnT (green) and visualized for tomato (red). Upper panels show GFP-injected heart in which tomato is seen only in fibroblasts. Lower panels show GHMT-injected heart in which tomato is seen in both fibroblasts and cardiomyocytes. Three types of iCLMs (positive for cTnT and tomato) were observed in injured hearts injected with GHMT-virus: (cell A) small cells without sarcomeres; cell B displays sarcomeres and expresses equivalent cTnT to neighboring tomato-negative cardiomyocytes; cell C displays sarcomeres and expresses less cTnT than neighboring tomato-negative cardiomyocytes. Scale bars, 20 μm. b. Tomato⁺ iCLMs isolated from tamoxifen treated Tcf21^iCre/Rosa26R^tdT mice one month post-MI followed by injection of GHMT retroviruses. Scale bar, 20 μm. c. Quantification of tomato⁺ cardiomyocytes isolated from uninjured hearts (n=2), injured hearts treated with empty vector retroviruses (n=3), or GHMT retroviruses (n=3). Data are presented as mean ± std. d. Recording of action potential of tomato⁺ iCLMs and endogenous cardiomyocytes by patch-clamping. Action potentials were recorded in response to brief (1–2 ms) depolarizing current (1–2 nA) injections delivered at 1 Hz by whole-cell current patch-clamping. (n=5 for each group).

Figure 4. Attenuation of cardiac function of injured hearts by GHMT

a. Cardiac function of mice subjected to LAD ligation followed by intramyocardial injection of GFP or GHMT retroviruses was evaluated at various time points by echocardiography. Data are presented as mean ± std. *: p<0.05, **: p<0.005, ns: not statistically significant. b. Cardiac function at 6 and 12 weeks post-MI was assessed by MRI. Data are presented as mean ± std. *: p<0.05, **: p<0.005.

Figure 5. Attenuation of fibrosis in response to MI by GHMT

a. Comparison of cardiac fibrosis and scar formation between GFP and GHMT infected myocardium 4 weeks after LAD ligation. Cardiac fibrosis was evaluated at 5 levels (L1–L5) by trichrome staining 4 weeks post-MI. The ligation site is marked as X. Severity of cardiac fibrosis was classified as mild, moderate or severe (fibrotic area <20%, 20–40% or >40%, respectively). Numbers indicate the various type of severity/total number of hearts examined in each group. A graph shows animal distribution in each category. Scale bar, 1mm. b. Quantification of fibrotic area in heart sections displayed in (a). Fibrotic area (%) = (the sum of fibrotic area at levels 3 and 4/the sum of myocardial area in the LV at levels 3 and 4) × 100. Data are presented as mean ± std. *: p<0.05, **: p<0.005.