Researchers have found definitive proof that a protein called titin acts as a “ruler” for thick filaments, the proteins that make muscles contract.
Throughout the muscles of the heart and body, the thick filaments have precise, uniform lengths. Scientists have long speculated that titin measures thick filaments, but no one, until now, had been able to provide evidence to support their theories.
“Functionally and clinically, it is very important to regulate the thick filament precisely, otherwise muscles would not function well,” says Henk Granzier, senior author of the study and professor of molecular and cellular medicine at the University of Arizona. “Biologists have always wondered what makes them so precisely structured.”
When titin breaks
Studying mice with certain mutations in the gene encoding the blueprint for the titin protein, the researchers found that when titin was shorter, so were the thick filaments, resulting in weakened muscles and dilated cardiomyopathy, a condition that leads to heart failure.
“We genetically engineered a mouse that doesn’t have normal titin,” says Granzier. “It has a piece from titin deleted. Since titin is involved in somehow regulating the thick filament, then you expect if you make titin shorter, the thick filament length will be altered as well. And, lo and behold, that is the case.”
“You might say titin rules…”
A typical protein is made of a few hundred amino acids linked in a chain. Though still microscopically small, titin is gigantic compared to other proteins. Comprised of more than 30,000 amino acids, the supersize protein is made from super-repeated structures. The amino acid super-repeats of titin are like tick marks on a yardstick, measuring out uniform sections of the thick filament.
By altering the gene for titin, Granzier’s team was able to make a mouse whose titin was missing several of its super-repeats.
The resulting mice showed symptoms of dilated cardiomyopathy, or DCM. This condition stretches out the muscle in the heart and prevents it from pumping efficiently. While the heart still contracts, the muscle is weak, so each contraction only moves a fraction of the blood pumped by a normal heart.
In humans, the most frequent cause of DCM is a mutation in titin that shortens the protein. DCM affects 1 in 500 people, and often patients must undergo a heart transplant to survive. Understanding the cause of the condition can better arm researchers as they search for novel ways to combat it.
Closing in on the answer
“In science, if you want to do conclusive work, you have to test your hypothesis multiple ways and get consistent results,” Granzier says. This meant that any abnormalities the researchers detected in the genetically engineered mice had to be deeply investigated.
After testing the strength of the mice, his team removed the muscles of interest to inspect them in a setting they controlled, instead of a setting controlled by the complex biological systems of the mice.
To make certain that differences in the mice were caused by titin, the muscle tissues were observed in vitro by removing them from the animals and artificially stimulating them to show that they produced less force. In the body, muscles contract when activated by calcium; under the microscope, flooding the isolated muscle tissue with a calcium solution mimics this activation process.
Using ultrasound, the team showed that the hearts of the engineered mice were larger than those of mice with normal titin. The next step was to investigate how this enlargement weakened the heart.
Titin and strength
“The sarcomere is the smallest muscle unit you can tease out and still have all the properties of muscle: force development and shortening,” Granzier says.
The sarcomeres are linked end-to-end in a chain that spans the entirety of a muscle. Just as a strong chain is made from links that are well made and uniform, sarcomere health and uniformity is vital to muscle strength.
Three filaments comprise each sarcomere: thick, thin, and titin filaments. The thick filament holds the motor driving contraction—myosin—at precise points, and it pulls the thick filament across the thin filament, causing muscle contraction.
Muscles are strongest when the thick and thin filaments overlap at an optimal length—around 2 micrometers, or one ten-thousandth of an inch. Stretch out the muscle, and the filaments cannot reach the point of optimum overlap, so the muscle is weakened. Overstretch the muscle, and the filaments do not overlap at all; the muscle cannot exert any force.
When titin is mutated and short, the resulting shortened fibers cannot reach the optimal point of overlap, and the muscle cannot exert much force. Shorter thick filaments cannot hold the optimal number of myosin motors, making the muscle even weaker. The thin and thick filaments do not overlap properly nor contract effectively when the thick filament is short.
Paula Tonino and Balazs Kiss, lead authors of the study and scientific investigators in Granzier’s lab, observed the muscle fibers under electron and super-resolution microscopes. They determined that in the muscles of engineered mice, not only were the thick filaments shortened, but also that they were shortened by precise, uniform lengths that corresponded to the size of the super-repeats removed from titin.
“We showed that titin is the regulator of the thick filament,” Granzier says, confirming that titin determines the strength of muscles and health of hearts.
“You might say titin rules,” he adds.
The researchers report their findings in the journal Nature Communications.
Source: Emily Walla for University of Arizona