Three years ago, Christine Seidman, MD, Director of the Cardiovascular Genetics Center, and colleagues were the first to sequence the behemoth titin gene responsible for encoding the largest protein in the human body. They discovered that mutations truncating titin are the most common genetic cause of severe and familial dilated cardiomyopathy (DCM) (N Engl J Med 2012;366:619-28).
Earlier this year, they expanded these studies to 5,000 individuals with a spectrum of cardiovascular physiology. They showed that titin mutations are the most common genetic cause for DCM in ambulatory patients and identified clinically important manifestations and outcomes for patients with titin mutations (Sci Trans Med. 2015; 14:270). They discovered how titin mutations do their damage to the heart muscle. Truncating titin mutations prevent development of the normal cardiomyocyte structure and impair the cardiomyocyte’s contractile performance (Science 2015; 349:982 -86).
Using next-generation sequencing, Dr. Seidman and colleagues sequenced the enormous titin gene in more than 5,000 individuals, including over 600 patients with dilated cardiomyopathy, over 3,000 community-based participants in the Framingham and Jackson Heart Studies with longitudinal cardiovascular data, and over 300 healthy controls. The researchers correlated the presence of mutations that truncated titin with the clinical manifestations in each cohort.
“We found that truncating titin mutations were present in 20 percent of patients with severe and in 13 percent with mild dilated cardiomyopathy,” said Dr. Seidman. These mutations were associated with marked reductions in the contractile function of the heart and increased the risk for arrhythmias. “We were also able to reveal that the location of the mutation mattered. Only truncating mutations that are predominantly in the titin A-band region were strongly associated with dilated cardiomyopathy,” added Dr. Seidman, “whereas other mutations, particularly those in the I-band, did not cause disease.”
To understand the different consequences in titin mutations, the laboratory characterized titin expression in human heart tissues. These studies showed that titin molecules in human hearts universally incorporated A-band sequences, but only variably incorporated I-band sequences. A mutation that altered sequences that are rarely expressed in the human heart would have little or any adverse consequences on contractile function.
“These findings have improved the accuracy of clinical genetic tests, which allows these to accurately screen relatives at risk for dilated cardiomyopathy. In addition, establishing genotype-phenotype correlation in the management of patients’ dilated cardiomyopathy due to titin mutations can be improved by early surveillance and interventions,” explained Dr. Seidman.”
To determine how truncated titin does its damage, Dr. Seidman and colleagues produced a cellular model of dilated cardiomyopathy – cell cultures of human cardiomyocytes with titin mutations that are derived from induced pluripotent stem cells. With collaborators in Dr. Christopher Chen’s laboratory at Boston University these cardiomyocytes were used to develop micro-cardiac tissues that contract, produce force, and respond to stimuli.
Since titin is known to be responsible for sensing and responding to myocardial stresses, Dr. Seidman and her colleagues suspected that titin truncating variants might exhibit aberrant stress responses. “We found that micro-tissues made from cardiomyocytes with titin mutations were less able to respond to mechanical and beta-adrenergic stress and had impaired growth factor and cell signaling activation,” she said. “These deficits are expected to impair cardiac adaptation to increased mechanical load and stress signals.”
The team also studied titin protein in cultures of human cardiomyocytes with titin truncating mutations. Some produced a stable truncated protein, a surprising observation. However, these mutant proteins did not assemble with other contractile proteins into well-organized functional sarcomeres. “The sarcomere is the contractile unit of muscle cells. Poorly organized sarcomere could account for basal contractile deficits and attenuated signaling,” explained Dr. Seidman.
RNA-sequence analyses also indicated that titin truncating mutations affected cardiomyocyte signaling and RNA expression. Notably there was diminished expression of several growth factors. “Interestingly, the growth factor deficit could be partially overcome by supplementing the cardiomyocytes with the vascular endothelial growth factor (VEGF),” she said.
“Based on this research,” Dr. Seidman said, “we can explain why patients with specific titin truncating mutations develop dilated cardiomyopathy and we now have some clues about potential therapeutic approaches to help limit the disease and its progression to heart failure.” With titin mutation as a target of therapy, pharmacologic agents could be developed to enhance titin gene expression, increase sarcomere formation, or stimulate cardiomyocyte signals that improve function.
The Cardiovascular Genetics Center at BWH is composed of a multidisciplinary team of internationally recognized physicians and investigators who collaborate to apply the latest discoveries in research on inherited cardiac disease to deliver personalized care for patients.
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