In response to repeated intense contractile activity skeletal muscle transiently loses its ability to generate force and motion. Despite extensive study the underlying molecular causes of this phenomenon remain unclear. Classic early studies suggested that the failure in this type of fatigue lay within the muscle. Since force and motion is ultimately generated by myosin binding to and translocating actin we have focused on understanding how these events might be perturbed during fatigue. Our approach is to assess the impact of putative fatigue agents on the molecular mechanics and kinetics of myosin using both the in vitro motility and laser trap assays.
Many of the putative causes of muscular fatigue are also thought to contribute to the rapid loss in cardiac contractility during acute myocardial ischemia. Therefore we are also interested in understanding the molecular mechanisms underlying this loss of function. In a current approach we are tackling this issue by increasing the complexity of our in vitro assays with the incorporation of the regulatory proteins troponin (Tn) and tropomyosin (Tm), in addition to actin. These actin-binding proteins indirectly affect the actomyosin interaction by regulating the availability of myosin binding sites on actin in a calcium dependent manner. The function of these regulatory proteins is thought to be disrupted during ischemia, but functional alterations and structures involved have not yet been fully characterized. A comparison of these results with those obtained in the absence of Tn/Tm will enable us to delineate the direct effects of Pi and H+ on actomyosin from indirect effects mediated through Tn/Tm.
Genetic cardiomyopathies often result from single point mutations to the contractile proteins in the myocardium, including myosin and actin as well as troponin and tropomyosin. These rather minute changes to the structure can cause gross pathological enlargement of the heart and ultimately cause it to fail. While it is clear that the presence of the mutation is the primary cause of the disease, the progression from mutation to pathology remains unclear. There are now over 100 identified mutations in sarcomeric proteins that are linked to cardiomyopathies that generally fall into two classes based on the phenotype. One class of mutations is associated with a thickening of the walls of the heart, known as hypertrophic cardiomyopathy (HCM). The second class of mutations causes the heart to enlarge due to a dilation of the chambers, known as dilated cardiomyopathy (DCM). My working hypothesis has been that these subtle changes in structure significantly alter function and it is this change in function that initiates the cascade of events that leads to the pathology. To test this hypothesis we have been comparing and contrasting the impact of both HCM- and DCM-causing mutations on the function of different contractile proteins in vitro.
In a new, but related, area of interest, my lab is beginning to investigate a third form of heart failure in collaboration with Dr. Tim Mader, an emergency room physician at Baystate Medical Center. This type of heart failure occurs when organized cardiac contractility abruptly stops and effective circulation ceases, a global form of ischemia. Even if a normal cardiac rhythm is rapidly restored (i.e. resuscitation) the profound global ischemia that occurs during the cardiac arrest and subsequent reperfusion injury often damages the myocardium (as well as other major organs) leading to what is known as post-cardiac arrest syndrome. The level of myocardial dysfunction following full resuscitation is a key component of this post-cardiac arrest syndrome and a significant contributor to morbidity and mortality, however the molecular basis of these effects remain poorly understood. Importantly, the dysfunction even occurs in the absence of pronounced necrosis and there is strong evidence to indicate that structural modifications might occur to the contractile proteins during the injury phase. We hypothesize that there will be detectable differences in both the structure and function of the contractile proteins and that these changes may underlie the myocardial dysfunction. Our approach to testing this hypothesis is to determine if the function of the contractile proteins is modified in our in vitro assays. These data will be combined with measures of whole heart function to enable us to link the molecular effects with in vivo function.