Heart disease remains the number one cause of mortality in the United States. Heart failure, a type of heart disease where the heart becomes weak and incapable of pumping blood sufficiently, causes significant morbidity and mortality. We have a better understanding of this disease thanks to the research performed in the past few decades; however, it is not yet complete. Therefore, it is essential to perform further research in order to better understand how the heart regulates its contraction during health and what goes wrong during heart failure.
Cardiac contraction is dependent on the cyclical interactions of the myosin heads with the actin filaments. This interaction drives force production and shortening, which result in pumping of the blood. The rate of this cycle, termed cross-bridge cycling rate, is an important determinant of cardiac output. Therefore, we set out to determine how this rate is regulated and disrupted in heart failure. Most of the previous studies on this parameter have used permeabilized cardiac preparations. This is a very reliable and established technique for assessing the function of the myofilaments. However, the cells in this type of preparation are de-membraned and experiments are conducted in essentially “non-living” cardiomyocytes. Furthermore, experiments are typically performed at sub-physiological temperatures. Therefore, we first developed a technique where cross-bridge cycling kinetics can be assessed in intact cardiac trabeculae under near-physiological conditions and body temperature (37 °C). By combining K+ contracture and rate of tension redevelopment (ktr) protocols, we were able to reproducibly assess cross-bridge cycling rate in intact cardiac trabeculae.
One of the mechanisms that the heart uses for regulating its contraction is the well established length-tension relationship where an increase in muscle length results in an increase in cardiac contractile tension. Some studies also show that muscle length affects cross-bridge cycling kinetics while others show lack of this relationship. These conclusions were drawn from experiments that were performed on permeabilized muscle preparations, at sub-physiological temperatures, or both. Therefore, we used our technique in order to assess the effects of muscle length on cross-bridge cycling kinetics under more relevant physiological settings. We demonstrated that cross-bridge cycling kinetics slow down as muscles are stretched in rat myocardium. This length-dependent regulation of cross-bridge cycling in rats does not necessarily mean that it also has relevance to humans. Due to the differences between the hearts of rats and humans, it was necessary to extend our studies to freshly isolated human myocardium.
Our experiments in the right ventricles of non-failing human myocardium showed a similar regulation. An increase in muscle length resulted in a decrease in cross-bridge cycling kinetics. We also performed experiments in the right ventricles of patients with end-stage heart failure, and observed a similar length-dependent regulation of cross-bridge cycling kinetics in these samples. Interestingly, the measured ktr values at each muscle length were similar between non-failing and failing human hearts. In order to complement these ktr studies, we also analyzed the parameters of twitch contraction. These measurements paralleled our ktr data in regards to both the effects of muscle length on kinetics, and comparison between non-failing and failing myocardium. These results collectively show that muscle length is capable of regulating cross-bridge cycling kinetics in both non-failing and failing human myocardium when assessed in intact muscle preparations under near-physiological conditions.
It has previously been shown that muscle length is a regulator of myofilament protein phosphorylation, most notably Myosin Light Chain-2 and Troponin I. Phosphorylation of both of these proteins have been implicated in determining cross-bridge cycling kinetics. Therefore, it is plausible that muscle length regulates cross-bridge cycling kinetics, in part, via phosphorylation of contractile proteins. Our preliminary results, however, did not identify a role of phosphorylation modifications in the length-dependent regulation of cross-bridge cycling kinetics. Additionally, during the course of our studies, we observed that tension transiently overshoots in the human ktr tracings. These overshoots have been previously documented and investigated in other types of muscles, preparations, and species; however, none exist for intact human cardiac muscles. Therefore, we performed a series of experiments which showed that overshoots in human cardiac muscles are most likely mediated by the cross-bridges.
During the course of our studies, we also documented that twitch tensions declined considerably, in contrast to the contracture tensions, over long periods of experimentation. We determined that a decrease in sarcoplasmic calcium reticulum content is, in part, responsible for the decline in twitch tension in rat myocardium. Additional studies led us to demonstrate that the sarcoplasmic reticulum calcium content during baseline stimulation frequencies is an important determinant of the relative force-frequency relationship. Decreasing this calcium content, results in an increase in the force-frequency relationship in rat myocardium.
The results of the experiments discussed in this work show that muscle length is an important factor in determining cross-bridge cycling rate of not only small rodents but more importantly, both non-failing and failing human myocardium. This research is, in part, novel in that all experiments used for reaching this conclusion were conducted in intact “living” muscles and at body temperature. This allows us to better understand how the heart regulates its contraction in health and disease.