Researchers from the Intermountain Healthcare Heart Institute in Salt Lake City have identified new mutations in a gene commonly associated with non-ischemic dilated cardiomyopathy (NIDC), a disease that weakens the heart muscle, making it more difficult to adequately circulate blood to meet the body’s needs.
Patients with NIDC struggle because the heart’s ability to pump blood is decreased, as the heart’s main pumping chamber, the left ventricle, is enlarged and dilated.
Unlike other kinds of heart conditions, NIDC often isn’t related to or a symptom or sign of a known cardiovascular disease or disease risk factor. While researchers have known that genetic factors play a role in non-ischemic dilated cardiomyopathy, that role isn’t entirely known.
In a new study presented to heart experts from around the world, researchers from the Intermountain Healthcare Heart Institute have identified 22 mutations in 27 of 229 NIDC patients in a gene called TITIN—15 of them not previously discovered.
These discoveries depended on applying advanced “whole exome sequencing”.
These TITIN mutations are of a type called “truncating variants”, or TTN-tv for short, which are linked with the development of cardiomyopathy and heart failure.
“Truncating mutations in TITIN are common in NIDC, so we wanted to know: if we find one, should we be more, or less worried about the patient’s prognosis?
The answer is yes,” said Jeffrey L. Anderson, principal investigator of the study and distinguished clinical and research physician at the Intermountain Healthcare Heart Institute.
In the study, the DNA samples of the 229 Intermountain patients diagnosed with NIDC were analyzed.
Researchers also identified lifestyle, environmental and other disease factors documented in the medical records that are associated with heart problems, like high blood pressure, diabetes, a history of alcohol or drug abuse, or previous chemotherapy treatment.
Patients were evaluated when they first presented and then were followed for five years.
Patients with a TTN-tv mutation more often had severe cardiomyopathy at presentation, and by five years they were less likely to have recovered (11% of those with a mutation vs. 30% of those without).
These patients also were more likely to have shown progressive disease, such as a heart transplant, implant of a permanent heart assist device, or death if they had a TTN-tv mutation (41%) than if they didn’t (25%).
TTN-tv mutation patients also commonly were found to have non-genetic predisposing factors, suggesting that these other factors may act in concert with genetic factors to precipitate heart failure.
Findings from the study were presented at the 2019 American Heart Association Scientific Sessions in Philadelphia on Nov. 17, 2019
“What we think is that, in many cases, people go along just fine with one of these mutations, but then other environmental and lifestyle or disease factors kick in, and it tips them over into non-ischemic dilated cardiomyopathy,” said Dr. Anderson.
“We think non-ischemic dilated cardiomyopathy is the result, in many cases, of a combination of genetic predisposing factors and environmental or other disease factors.”
“Currently, physicians don’t routinely test patients with NIDC for all of these known and new TTN-tv gene mutations, and there’s no specific genetic treatment for the disease,” said Dr. Anderson.
“However, if patients were to be tested and identified as having a disease-predisposing mutation, physicians could then be more aggressive about monitoring them and treating them with known heart failure drugs and devices,” he added.
“If we test patients for one of these disease-related mutations, we can identify them and also affected family members whose disease is more likely to occur and then progress so we can be better on top of prevention and treatment measures.”
ilated cardiomyopathy (DCM) occurs as many as 1 in 250 people1. There are currently no approved therapeutic products indicated for DCM treatment. Typical treatments are those indicated for broader cardiovascular disease. As the disease progresses, patients have limited treatment options, such as surgical or other invasive interventions and heart transplant2.
DCM will result in heart failure with reduced ejection fraction (EF), usually without prior ischemic. The walls of the left ventricle are thin and over-expanded, leading to improper contraction and insufficient blood being pumped by the dilated heart. DCM results from a variety of external factors, such as viral infection, alcohol abuse, exposure to cardiotoxic medications and pregnancy, as well as from genetic variants in a number of causal genes including TTN, LMNA, ACTC1, MHY7 and PLN3–9.
Titin (coded by TTN) plays an important role in the contraction and relaxation of cardiac muscles by connecting Z-disc to the M-line in the sarcomere. TTN truncating variants (TTNtv) contribute up to 15% ambulatory DCM and 25% end-stage or familial DCM3,8,9. In DCM, TTNtv are significantly enriched most in A band as well as other regions including I-band, Z-disc or M-line with variable position-related odds ratios3,10.
TTN haploinsufficiency caused by TTNtv is emerging as the potential disease mechanism. Rat models with TTNtv in Z-disc and A band did not result in change in titin protein levels and obvious cardiac performance under normal physiological conditions. It was not known whether Ttn insufficiency causes DCM in mouse.
Mammalian cardiomyocytes exit their cell cycle shortly after birth, preventing heart repair through cardiac regeneration11. Cell cycle reactivation is observed in a limited cardiomyocytes under physiological conditions12,13.
As an emerging strategy for cardiac therapeutic regeneration, we and others showed that enhancing this process by removing cell cycle brakes or augmenting accelerators is beneficial for heart recovery from heart failure models caused by ischemia or pressure overload14–18.
We previously observed that cardiomyocytes undergo an extra round of cell cycle in Mybpc3 deficient mice, suggesting cell cycle reactivation could compensate sarcomere insufficiency19.
It was not known whether Ttn insufficiency could induce cardiac cell cycle reactivation. Moreover, it was unknown whether enhancing this process could be a therapeutic strategy for DCM caused by Ttn insufficiency. Here, we address these major gaps and identify therapeutic candidates that are vital for advancing potential hits into a therapeutic approach for DCM.
Discussion
Ttn truncating variants are enriched in DCM patients suggesting a causal effect of TTN variants on DCM3. TTN Haploinsufficiency caused by TTNtv is emerging as the disease mechanism. To assess whether Ttn insufficiency causes DCM, we used shRNA to modulate Ttn expression. By reducing ~ 50% of Ttn expression, we generated a mouse model demonstrating a severe DCM phenotype, including ventricular wall thinning, dilated ventricular chambers and impaired cardiac function.
To develop potential therapy for suppressing DCM caused by Ttn insufficiency, we screened 10 genes involved in different pathways. As a transcriptional regulator of TTN during direct cardiac reprogramming, we included Yy1 for the rescue experiments. Ttn expression was not induced by Yy1 in DCM hearts caused by Ttn shRNA, suggesting that Yy1 does not inhibit DCM through Ttn gene regulation in mouse hearts. Recently, Yy1 was shown to promote both Nkx2.5 expression, cardiac progenitor cell commitment and maintenance during early embryo development34,35.
Cardiomyocyte-specific ablation of Yy1 mediated by Myh6-cre resulted in perinatal death of mutant mice, suggesting Yy1 plays an important role in early and late cardiac lineage development36. Importantly, YY1 is shown to be upregulated in human idiopathic dilated cardiomyopathy (IDC) and heart failure37,38.
We detected over 35 fold increase of Ccnd1 and Ccnd2 in cardiomyocyte nuclei upon Ttn shRNA, indicating cardiomyocytes response to Ttn insufficiency by activating cell cycle reentry signals. We did not observed this reaction in another DCM model caused by Lmna insufficiency. Activated cardiomyocyte cell cycle induced by Ttn insufficiency does not advance to S phase. We found that upregulation of Yy1 promoted cardiac cell cycle reentry by further enhancing Ccnd1 and Ccnd2. Yy1 promoted cardiomyocyte cell cycle to S phase by a significant increase of EdU incorporation. However, we did not detect mitotic phase marker pH3 in cardiomyocytes, suggesting activated cardiomyocytes undergoing mitotic phase are limited.
Importantly, Upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Taken together, Yy1 promotes cardiac cell cycle to facilitate to suppress Ttn shRNA-induced DCM.
Our recent study showed insufficiency of Mybpc3, another causal gene for sarcomeric cardiomyopathy, induces an extra round of cardiomyocyte cell cycle during neonatal stage19.
It is now of great interest to know whether this reactivation of cardiac cell cycle is a common mechanism for sarcomere deficiency and enhancing of this process by Yy1 or other regulators is able to suppress cardiovascular disease related to sarcomere. Reactivation of cardiomyocyte cell cycle does not always lead to cell number increase.
Cytokinesis is still rarely detected in adult hearts upon regenerative enhancement. Yy1 might promote hypertrophic growth coupled with cell cycle reentry as Ccnd2 is a mediator for Myc overexpression or exercise-induced hypertrophic growth in cardiomyocytes39. Recently, Yy1 is suggested to serve as a structural regulator between enhancer and promoter interactions and facilitates gene expression40.
Apart from modulating cell growth, upregulation of Yy1 might indeed reinstate or promote enhancer – promoter interaction to restore gene regulatory network dysregulated by Ttn insufficiency.
Our selected candidates included many therapeutic targets for heart failure including Atp2a2, Bcl2, Sod2 and Sirt323,24,28,41. Most of them failed to protect DCM induced by Ttn shRNA, suggesting root causes and disease mechanisms should be taken into account for DCM prevention and treatment strategies.
It is of great interest to know whether Yy1 is able to suppress DCM caused by other genes including LMNA, MYH7 and PLN. To translate our research, one concern is whether upregulation of Yy1 could cause any cardiac defects. Previous study showed overexpression of Yy1 induced a relative marginal hypertrophy cardiomyopathy only in male mice. Here, we specified the Yy1 expression by cardiac specific promoter, virus dose and postnatal transduction.
In contrast to a previous study, no detectable impairment of cardiac structure and performance was observed after ~ 5 months of virus transduction37. Taken together, our findings provide a strong supporting evidence for translational research.
Provided by Intermountain Medical Center
