Published in Scientia, May 17, 2017. Read the full article here.
Non-communicable diseases, such as heart disease, obesity, and diabetes, kill more people worldwide than any other disease. Drs Petra Kienesberger and Thomas Pulinilkunnil at Dalhousie University have dedicated their careers to understanding the molecular underpinnings of these diseases, in the hope of creating a healthier tomorrow.
Heart disease has been the leading cause of death worldwide for over 15 years, killing millions of people each year. Diabetes recently rose to the sixth leading cause of death worldwide, and is projected to move further up the list in the future. These two diseases are often associated and share risk factors, as well as distinct metabolic and molecular signatures. While scientists have a grasp on many factors that are associated with heart disease and diabetes, often the underlying mechanisms are not well understood, presenting a barrier to the development of novel treatments for these conditions. The cornerstone of Dr Kienesberger and Dr Pulinilkunnil’s research program is illuminating the role of cellular energy metabolism in governing outcomes of metabolic heart disease.
Autophagy, Obesity, and the Heart
Diabetes and obesity are often linked with heart disease. In particular, many diabetic and obese patients suffer from a group of conditions known as cardiomyopathy – weakening of the cardiac muscle. The progression of cardiomyopathy often leads to heart failure, thus an understanding of the factors that contribute to and predict cardiomyopathy may help doctors to develop new treatments to reduce and prevent heart disease related deaths in the future. Drs Kienesberger and Pulinilkunnil noted that cardiomyopathy in obese and diabetic patients is often associated with changes in cardiac metabolism that lead to glucolipotoxicity, an accumulation of toxic levels of glucose and fatty acids and their metabolites in the bloodstream and in the cardiac cells. When this occurs, cardiac cells start to struggle with energy regulation and stop disposing and recycling unneeded and damaged proteins, which can ultimately lead to cell death. The Pulinilkunnil laboratory hypothesised that glucolipotoxicity interferes with the cells’ natural waste disposal system and sought to identify and characterise the underlying mechanisms.
Cells get rid of waste, such as used proteins, via intracellular organelles called lysosomes. These tiny vessels engulf unwanted organic materials and digest them through a process known as autophagy. Through autophagy, the cell is able to break down and recycle cellular waste to provide energy for the cell and create fresh building blocks for new proteins and cellular products. When lysosomes are unable to perform autophagy, the cell begins to build up unwanted proteins and waste, which can damage the health of the cell. Lysosome autophagy is controlled by a suite of specialised signalling proteins called transcription factors. One of the most important of these proteins is master regulator transcription factor EB, or TFEB for short. TFEB signals for cells to produce new lysosomes, and coordinates the activity of active lysosomes to keep autophagy running smoothly in the cell. The team speculated that TFEB activity and lysosomal function were disrupted in glucolipotoxic conditions.
To test this, they started with observing the heart tissue of obese and diabetic mice. They found that TFEB was present in lower levels in diseased animals than in healthy controls. Next, they grew rodent cardiac cells in a petri dish, and exposed them to differing levels of glucose and fatty acids, allowing them to observe the response of the cells in detail. The isolated cells exposed to glucolipotoxic conditions expressed lower levels of TFEB and showed impaired lysosomal autophagy. Notably, a particular subset of fatty acids seemed to be most toxic, indicating that this mechanism could be targeted in obesity and diabetes to ameliorate cardiomyopathy. To test whether the rodent results held up in humans, small samples of heart tissue were collected from human patients undergoing heart surgery for various cardiac disorders. Analysis of this tissue revealed that indeed, morbidly obese patients with heart disease had lower levels of TFEB than non-obese patients. These results highlighted the connections between conditions and disease, and revealed impoverished TFEB as a potentially critical player in obesity and diabetes related heart disease.
The Complex Role of Fats in Heart Health and Disease
An interest in the consequences of cardiac cell metabolism and glucolipotoxicity led Dr Kienesberger and colleagues to review the role of lipids in cardiac disease, in an effort to tease out the most damaging fats. Cardiac lipotoxicity, the over-accumulation of specific lipids in heart tissues, often leads to damaged cardiac cells, insulin resistance, metabolic dysfunction, and ultimately cell death. However, there are many types of lipids, and how they interact with cardiac tissue is not well understood, leading to the classifications of ‘good’ and ‘bad’ fats. However, much of the research in this area has focused on a single lipid type, and may be neglecting the bigger picture as to how fat molecules and cardiac cells interact. Dr Kienesberger performed a comprehensive survey of the available research on lipotoxicity and found trends that may indicate a complex role for how lipids interact with cardiac tissue in the development of heart disease.
Heart tissue often prefers to consume fatty acids for energy, but healthy cardiac cells have a more flexible metabolism, consuming both fatty acids and sugars present in the blood stream to keep the heart pumping. Diseased hearts often start to show less metabolic flexibility, relying on fewer energy sources and making cells more vulnerable to impairment. Disease states are often associated with the build-up of fatty plaques in the heart tissue, a situation that occurs when there are more fatty acids available than the heart can metabolise. In studies focused on single types of fats, certain lipids appear to be more toxic than others when they build up in these plaques, while others have been labelled as harmless or ‘good’. Dr Kienesberger’s analysis indicates that the reality is much more complex. Some lipids that appear harmless on their own may have components that are toxic or interact with other molecule to cause damage. Dr Kienesberger argues that more comprehensive research on the networks of these molecular interactions is necessary to form a full picture of the processes that underlie lipotoxicity. However, a consistent finding across all studies is that limiting fatty acid uptake prevents the fatty overaccumulations that contribute to heart disease. Therapeutics that aim to limit fatty acid uptake may help prevent heart damage, regardless of lipid type.
A New Understanding of Chemotherapy Induced Heart Disease
The Pulinilkunnil laboratory further expanded this research to other models of heart failure commonly observed in drug induced cardiomyopathy – specifically that induced by Doxorubicin, a drug used for cancer treatment. Doxorubicin, or DOX, is an effective chemotherapy medication commonly used to treat many different kinds of cancer and notably breast cancer. Chemotherapies are often known for their unpleasant side effects such as hair loss and nausea, but DOX is also known to increase susceptibility to cardiomyopathy specifically in women, through mechanisms that are not fully understood. Prolonged treatment with DOX often leads to heart failure, requiring physicians to balance cancer treatment with heart disease risk. DOX is known to suppress mitochondrial metabolism, alter calcium levels, and interfere with protein break down in many kinds of cells, but its effects on cardiac cell waste processing has only recently been uncovered.
Prior work established that there is a perturbation of lysosomal autophagy activity in the cardiac muscle of DOX treated patients, but it was unclear how DOX might be influencing these processes. The Pulinilkunnil laboratory demonstrated that DOX repressed the expression of TFEB and reduced the activity of cathepsin B – a proteolytic enzyme essential for lysosomal autophagy. Overall, they found that the loss of TFEB reduced the expression of many proteins involved in autophagy, disrupted the normal flux of autophagy activities in the cells, reduced the availability and activity of cathepsin B, and increased the activation of cell death programs.
While DOX appears to disrupt mitochondrial activity, restoration of mitochondrial function has largely failed to prevent the drug’s negative effect on heart health. Dr Pulinilkunnil argues that a component of DOX’s apparent effect on mitochondrial activity is related to its influence on autophagy processes; cardiac cells possess a particularly high number of mitochondria, and when unable to recycle, defective mitochondria quickly become overloaded and unable to function. The Pulinilkunnil laboratory’s careful investigation of the DOX-influence pathways associated with lysosomes and autophagy reveal that the drug interferes with autophagic processes at multiple levels, and the disruption of these pathways is what ultimately results in cardiac damage.
In cardiac tissue, DOX interferes with autophagy by altering lysosome structure and impairing transcription factors that regulate lysosomal activity, including TFEB. This results in a build-up of waste in cardiac cells, creating a toxic environment inside the cell and leading to cell death, causing widespread cardiac tissue damage and eventually resulting in heart failure. Taken together, these effects can lead to widespread cardiac tissue damage, increasing the chances of heart complications following cancer treatment with DOX. Despite this grim result, the team found a silver lining: when TFEB was restored to the cells, many of these effects were reduced and cardiac cells were less likely to undergo programmed cell death. This highlights the potential for TFEB as a research target for preventative cardiac health treatment.
In this regard, the Pulinilkunnil laboratory recently published an in-depth review on this topic, detailing what is known about the molecular relationships between these components of health and illuminating novel pathways through which DOX influences heart health. Dr Pulinilkunnil has grown particularly fascinated with the connections between lysosomal autophagy and cardiomyopathy. Studying the effects of metabolites, drugs, peptides, novel compounds, dietary substances, nutrients on lysosomes and TFEB allows him to reveal the pathways involved in autophagy processes and better appreciate how these entities influence functional outcomes in healthy individuals, with the hope that this increased understanding will lead to advances in our understanding of metabolic heart disease and help discover new therapeutics to boost cardiac health down the line.
Tying Obesity to Disease
For both heart disease and diabetes, a high level of body fat is a risk factor, and excess adipose tissue plays a role in disease development. While these effects are routinely attributed to alterations in metabolism associated with obesity, the hormonal and molecular pathways that contribute to disease progression are not fully known. Autotaxin, or ATX, is a protein produced by adipose cells that is released into the blood stream. Once secreted, ATX produces a fat molecule that triggers important signalling cascades in many cell types of the body. ATX has been implicated in cardiovascular disease and, more recently, obesity and diabetes, but it is currently unclear whether it aggravates or mediates progression of obesity and diabetes. The Kienesberger laboratory sought to better understand the behaviour of this protein and its relationship with blood sugar regulation and metabolism.
The team began by examining ATX blood serum levels in mice that had been raised on either regular chow diet or a diet containing high fat and high sugar (HFHS), and had blood collected either closely following a meal or after a 16 hour fast. Mice on the HFHS diet were overweight and had higher levels of ATX. Mice that had fasted prior to blood collection had lower levels of ATX regardless of body condition. These results confirmed that ATX levels are related to overall fat accumulation, but may fluctuate with meals.
Next, the researchers grew adipose cells in petri dishes to more closely analyse ATX secretion in response to varying doses of glucose and insulin, which controls many of the body’s metabolic processes. They found that fat cells with induced insulin resistance due to their inability to properly respond to insulin showed increased ATX secretion, and this effect could be prevented by adding an insulin sensitiser to the mix. Glucose appeared to have a dose-dependent effect on ATX secretion overall – predictably increasing ATX levels as glucose levels went up. Insulin had a more nuanced effect – a short spike of insulin increased ATX, but chronic exposure to insulin decreased ATX levels. ATX levels appeared to be influenced by both short- and long-term changes in nutritional states, blood sugar levels and insulin.
Making Strides in Human Health
The team’s future research directions will continue to pursue a deeper understanding of human health. With the help of a grant from the Heart and Stroke Foundation of Canada – New Brunswick and the New Brunswick Health Research Foundation, Dr Kienesberger intends to delve deeper into the mysteries of ATX, further teasing apart the relationship between this elusive protein, diabetes, and heart health. Dr Pulinilkunnil recently received a grant from the Canadian Diabetes Association to study the role of TFEB and autophagy in mediating heart disease, with the hopes of developing novel treatments for cardiomyopathy.