lncExACT1 and DCHS2 Regulate Physiological and Pathological Cardiac Growth

Abstract

Background: The heart grows in response to pathological and physiological stimuli. The former often precedes cardiomyocyte loss and heart failure; the latter paradoxically protects the heart and enhances cardiomyogenesis. The mechanisms underlying these differences remain incompletely understood. Although long noncoding RNAs (lncRNAs) are important in cardiac development and disease, less is known about their roles in physiological hypertrophy or cardiomyogenesis.

Methods: RNA sequencing was applied to hearts from mice after 8 weeks of voluntary exercise-induced physiological hypertrophy and cardiomyogenesis or transverse aortic constriction for 2 or 8 weeks to induce pathological hypertrophy or heart failure. The top lncRNA candidate was overexpressed in hearts with adeno-associated virus vectors and inhibited with antisense locked nucleic acid-GapmeRs to examine its function. Downstream effectors were identified through promoter analyses and binding assays. The functional roles of a novel downstream effector, dachsous cadherin-related 2 (DCHS2), were examined through transgenic overexpression in zebrafish and cardiac-specific deletion in Cas9-knockin mice.

Results: We identified exercise-regulated cardiac lncRNAs, called lncExACTs. lncExACT1 was evolutionarily conserved and decreased in exercised hearts but increased in human and experimental heart failure. Cardiac lncExACT1 overexpression caused pathological hypertrophy and heart failure; lncExACT1 inhibition induced physiological hypertrophy and cardiomyogenesis, protecting against cardiac fibrosis and dysfunction. lncExACT1 functioned by regulating microRNA-222, calcineurin signaling, and Hippo/Yap1 signaling through DCHS2. Cardiomyocyte DCHS2 overexpression in zebrafish induced pathological hypertrophy and impaired cardiac regeneration, promoting scarring after injury. In contrast, murine DCHS2 deletion induced physiological hypertrophy and promoted cardiomyogenesis.

Conclusions: These studies identify lncExACT1-DCHS2 as a novel pathway regulating cardiac hypertrophy and cardiomyogenesis. lncExACT1-DCHS2 acts as a master switch toggling the heart between physiological and pathological growth to determine functional outcomes, providing a potentially tractable therapeutic target for harnessing the beneficial effects of exercise.

Keywords: Hippo signaling pathway; RNA, long noncoding; Yap1 protein, human; exercise; heart failure; hypertrophy.

Figure 1.. Identification of exercise-associated cardiac lncRNAs (lncExACTs).

A. Volcano plot showing lncRNA RNAseq results in hearts from exercised compared with sedentary mice, n=4 mice/group. B. Venn diagram of differentially regulated cardiac lncRNAs in exercised (Run) mice and animals with transverse aortic constriction (TAC)-induced left ventricular hypertrophy (TAC-LVH) or heart failure (TAC-HF), n=4 mice/group. C. QRT-PCR measurement of cardiac lncExACT1–5 expression in control (Ctrl), exercised (Run), TAC-LVH, or TAC-HF mice. *p<0.05 vs. Ctrl by one-way analysis of variance (ANOVA) with post hoc Tukey, n=4–5 mice/group. D. lncExACT1 expression measured by QRT-PCR is increased in hearts from heart failure (HF) patients compared with controls, n=12/group. p<0.001 by Student’s t test. E. Plasma lncExACT1 determined by ddPCR is increased in patients with HF with reduced ejection fraction (HFrEF, n=18) or HF with preserved ejection fraction (HFpEF, n=16) in comparison to supraventricular tachycardiac (SVT) patients without HF (n=8). p=0.006 and p=0.032 by pairwise Wilcoxon rank sum test with Bonferroni correction. Data shown as mean±SEM.

Figure 2.. lncExACT1 overexpression induces pathological hypertrophy.

A. QRT-PCR measurement of lncExACT1 in mouse hearts 16 weeks after injection with AAV-lncExACT1 (lncExACT1) or AAV-GFP (Con). B Heart weight (HW) relative to tibial length (TL). C. Relative wall thickness (RWT). D. Fractional shortening (FS). E. Lung weight (LW) relative to tibial length (TL). F. Left ventricular end diastolic internal dimension (LVIDd). G. QRT-PCR measurement of hypertrophy markers in the heart. H. Quantification of cardiomyocyte area from wheat germ agglutinin (WGA)-stained heart sections. I. Quantification of EdU, PCM1 double-positive cardiomyocytes in heart sections. *p<0.05, **p<0.01 by Student’s t test. Data shown as mean±SEM.

Figure 3.. lncExACT1 inhibition induces physiological hypertrophy.

A. QRT-PCR measurement of lncExACT1 in hearts from mice injected with LNA-GapmeR-Control (Con) or LNA-GapmeR-lncExACT1 (Gap) for 2 weeks. B. Heart weight (HW) relative to tibial length (TL). C. Relative wall thickness (RWT). D. Fractional shortening (FS). E. Lung weight (LW) relative to tibial length (TL). F. Left ventricular end diastolic internal dimension (LVIDd). G. QRT-PCR measurement of hypertrophy markers in the heart. H. Quantification of cardiomyocyte area from wheat germ agglutinin (WGA)-stained heart sections. I. Quantification of EdU, PCM1 double-positive cardiomyocytes in stained heart sections. J. Quantification of Ki67, PCM1 double-positive cardiomyocytes in stained heart sections. K. Quantification of pHH3, PCM1 double-positive cardiomyocytes in stained heart sections. *p<0.05, **p<0.01 by Student’s t test. Data shown as mean±SEM.

Figure 4.. lncExACT1 inhibition protects against pathological hypertrophy, cardiac dysfunction, and fibrosis.

A. QRT-PCR measurement of lncExACT1 expression in hearts from mice subjected to sham operation (Sham), transverse aortic constriction with LNA-GapmeR-Control injection (TAC), or TAC with LNA-GapmeR-lncExACT1 (TAC+Gap). B Heart weight (HW) relative to tibial length (TL). C. Fractional shortening (FS). D. Left ventricular end diastolic internal dimension (LVIDd). E. Relative wall thickness (RWT). F. QRT-PCR measurement of hypertrophy markers in the heart. G. Quantification of fibrotic area from Masson trichrome-stained heart sections. H. Quantification of cardiomyocyte area from heart sections with wheat germ agglutinin (WGA) staining. I. Quantification of Ki67 and pHH3, and PCM1 double-positive cardiomyocytes in heart sections. J. QRT-PCR measurement of lncExACT1 expression in hearts from mice subjected to sham operation (Sham), myocardial ischemia reperfusion (IR) with LNA-GapmeR-control injection, or IR with LNA-GapmeR-lncExACT1 (IR+Gap). K. FS. L. Quantification of EdU and pHH3, and PCM1 double-positive cardiomyocytes at the infarct border zone in heart sections. M. Quantification of fibrotic area in Masson trichrome-stained heart sections. N. Quantification of TUNEL and cardiac troponin I (cTnI) double positive cardiomyocyte in heart sections. *p<0.05, **p<0.01; in I *p<0.05 vs. Sham, #p<0.05 vs. TAC by one-way analysis of variance (ANOVA) with post hoc Tukey. In K, *p<0.05 vs. Sham, #p<0.05 vs. IR+Gap by repeated repeated measures ANOVA. Data shown as mean±SEM.

Figure 5.. lncExACT1 works through DCHS2.

A. QRT-PCR measurement of DCHS2 in hearts from HF patients and controls (n=12/group), and in mice with HF or lncExACT1 overexpression, or exercise (Run) or lncExACT1 inhibition (Gap). B. Luciferase activity driven by DCHS2 promotor fragments in NRVMs with lncExACT1 OE or KD. C. QRT-PCR measurement with primers targeting different regions of DCHS2 promoter in complex from pulldown with probes targeting sense and antisense sequence of lncExACT1. D. Quantification of EdU and troponin double-positive cardiomyocytes by flow cytometry. E. Quantification of cardiomyocyte size in NRVMs treated with scrambled control or DCHS2 siRNA. F. QRT-PCR measurement of hypertrophy gene markers, n=3/group. G. Quantification of EdU positive cardiomyocytes by flow cytometry. H. Quantification of cardiomyocytes size after lncExACT1 OE and/or DCHS2 KD in NRVMs. *p<0.05, **p<0.01; in C and D, *p<0.05 vs. Control, #p<0.05 vs. lncExACT1 KD. A, C, D, and E by Student’s t test; B, F, G, and H by one-way analysis of variance (ANOVA) with post hoc Tukey. Data shown as mean±SEM.

Figure 6.. DCHS2 overexpression induces pathological cardiac hypertrophy and reduces regenerative capacity in zebrafish.

A. Representative image from apex region of an adult transgenic zebrafish heart immunostained for tropomyosin (Red), GFP (Green), and nuclei (DPAI, Blue). B. Representative image and quantification of cardiomyocytes size isolated from wild-type control (CTRL, white arrow) or DCHS2 overexpression (hGFP-DCHS2, yellow arrow) zebrafishes. C. QRT-PCR measurement of ANP and BNP in hearts from CTRL and hGFP-DCHS2 zebrafishes. D. Representative images and quantification of nkx2.5 and PCNA double positive cardiomyocytes in hearts at 7 days post injury (dpi) from CTRL and hGFP-DCHS2 zebrafish. E. Representative images and quantification of fibrosis in hearts at 60 days post injury (dpi) from CTRL and hGFP-DCHS2 zebrafish. F and G. Representative images and quantification of nuclear total Yap1 and cytoplasmic p-Yap1 protein expression in hearts from CTRL and hGFP-DCHS2 zebrafish. *p<0.05, **p<0.01 by Student’s t test. Data shown as mean±SEM.

Figure 7.. DCHS2 knockdown promotes physiological cardiac hypertrophy.

A. Schematic of experimental strategy employed to knockdown DCHS2 in the hearts in vivo. B. QRT-PCR measurement of cardiac DCHS2 in Cas9 knockin mice with injection of AAV9 carrying gRNA1 or gRNA2. C. Heart weight (HW) relative to tibial length (TL). D. Quantification of cardiomyocyte area from wheat germ agglutinin (WGA)-stained heart sections. E. Lung weight (LW) relative to tibial length (TL). F. Fractional shortening (FS). G. Relative wall thickness (RWT). H. Left ventricular end diastolic internal dimension (LVIDd). I. QRT-PCR measurement of hypertrophy gene markers. J. Quantification of Ki67. K. Quantification of pHH3, PCM1 double-positive cardiomyocytes in stained heart sections. L and M. Representative images and quantification of nuclear total Yap1, and cytoplasmic p-Yap1, p-MST1/2, and total MST1 (t-MST1) protein expressions in hearts from mice injected with AAV9 carrying control, or gRNA1, or gRNA2. *p<0.05 by one-way analysis of variance (ANOVA) with post hoc Tukey. Data shown as mean±SEM.