Kenneth Campbell, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Diastolic dysfunction is the most common cause of congestive heart failure in elderly populations. One year mortality rates for afflicted individuals aged 70 years or older approximate 50%. Therapy options remain empirical. Although very little is known about the etiology of Diastolic Heart Failure (DHF) most studies agree that affected patients exhibit increased ventricular stiffness (reduced chamber compliance). The hypothesis underlying this work is that DHF in elderly populations reflects cross-bridge activity that persists inappropriately during the diastolic (or 'relaxed') phase of the cardiac cycle. These cross-bridges produce an 'active' component of myocardial stiffness that augments the heart's basal (passive) stiffness and impairs ventricular filling by increasing the resistance to inflowing blood. The proposed research will utilize Fischer 344 rats that exhibit aging-associated DHF at 25 months of age. Specific Aim 1 will establish the effects of aging on rat myocardial stiffness. Experiments will test the hypothesis that active stiffness due to inappropriately bound cross-bridges increases to a greater extent with aging than stiffness due to structural components. Intact trabeculae will be isolated from young (5 month) and old (25 month) rats and stretched in the presence and absence of BDM, a cross-bridge inhibitor, to establish the extent of age-dependent changes in active and passive stiffness. Specific Aim 2 will evaluate the effects of altered metabolite concentrations on active stiffness in young and old hearts. Experiments will utilize chemically permeabilized preparations isolated from 5 month and 25 month rat hearts and active stiffness will be assessed by measuring the tension responses to small stretches imposed under sarcomere length control. It is hypothesized that active stiffness due to persistent cross-bridge activity will be enhanced to a greater extent in the old hearts than in the young hearts when the concentrations of hydrogen ions, phosphate ions and ADP are raised to levels mimicking ischemic myocardium. This research is relevant to public health because it investigates the novel hypothesis that aging-related DHF reflects inappropriate contractile activity during the relaxed phase of the cardiac cycle. The experimental results will provide new information about the underlying causes of DHF and should help scientists develop better treatments for diastolic dysfunction in elderly populations. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Diastolic heart failure is a major cause of illness and death. The condition is nearly always associated with increased ventricular stiffness and is more common in elderly and/or obese populations. There are no effective clinical treatments. The central hypothesis underlying this research is that diastolic myocardial stiffness is the sum of an 'active'stiffness component (due to myosin heads that continue to cycle during diastole) and a 'passive'stiffness component (attributed to titin, collagen, elastin and intermediate filaments). The proposed research determines the relative contributions from these components in preparations ranging from single myocytes to whole hearts and uses these results to create a predictive computational model of diastolic stiffness. Specific Aim 1 will identify the molecular components responsible for the increased stiffness of myocardium from aged rats. The working hypothesis is that the increased stiffness reflects slowed acto-myosin kinetics. Experiments will measure the mechanical properties of myocardium from 6, 18, 22 and 26-month-old Fischer 344 rats by subjecting chemically permeabilized single myocytes and multicellular preparations to repeated stretches at different levels of calcium activation. Additional experiments will measure ventricular stiffness in the different aged animals by rapidly inflating balloons placed inside the left ventricles of Langendorff-perfused hearts. Titin and myosin isoform content will be measured by gel electrophoresis. Collagen and elastin content and collagen cross-linking will be determined using histological and biochemical techniques. Specific Aim 2 will use identical methods to identify the molecular components responsible for the increased myocardial stiffness evident in a rat model of diet-induced obesity. The working hypothesis for this aim is that the elevated myocardial stiffness observed in obese Sprague-Dawley rats reflects increased collagen content and/or collagen cross-linking. Specific Aim 3 uses the experimental results from Aims 1 and 2 to create a predictive computational model of diastolic stiffness. The model framework will consist of elastic and visco-elastic elements (representing titin, collagen, elastin and intermediate filaments) arranged in parallel with a spatially-explicit simulation of acto-myosin interactions. Model parameters will be determined by multi-dimensional optimization. The final model will be used to test predictions about the cross-bridge component of myocardial stiffness and should prove useful for assessing the likely effects of potential new treatments for diastolic heart failure. NARRATIVE This research is relevant to public health because it seeks to identify why hearts become excessively stiff in a common cardiovascular disease called Diastolic Heart Failure. The experimental results should help scientists to develop better treatments for the disease in overweight and elderly patients.

ABSTRACT The goal of this research is to develop a predictive multiscale model that will improve understanding of familial cardiomyopathies and that can be used to help screen potential new therapies for cardiac disease. Familial cardiomyopathies are the most frequently inherited heart defect and affect about 700,000 Americans. Most of the genetic mutations affect myosin or regulatory proteins that modulate myosin function. The majority of these mutations also induce abnormal cardiac growth termed hypertrophy. This project will develop, calibrate, and validate an innovative multiscale model that uses data quantifying myosin-level function to predict how hearts hypertrophy over time. This is a critical step on the path to developing patient-specific computer models that can be used to optimize treatments for heart failure and to predict the effects of different types of pharmaceutical intervention. In the future, one could envision clinicians testing drug treatments in silico and selecting the intervention that produces the greatest long-term benefit for their patient. The research team consists of two physiologists/biophysicists (Campbell & Yengo) and two engineers (Wenk & Lee) who share a common interest in cardiac biology. Together, their research skills span from structure-function analysis of myosin molecules to computer simulations of hearts that grow and remodel over time. The research plan integrates state-of-the-art hierarchically-coupled mathematical models with validation experiments that range from stopped-flow molecular kinetic assays to magnetic resonance imaging of myocardial strain patterns. The model will be tested using molecular to organ-level experimental data obtained from wild-type mice and from transgenic animals that develop cardiac hypertrophy because of a K104E mutation in myosin regulatory light chain. Additional tests will be performed using drugs that enhance (omecamtiv mecarbil) and inhibit (MYK-461) myosin-level contractile function. There are three specific aims. Aim 1: Integrate a multistate kinetic model of myosin into an organ-level finite framework to predict the effects of genetic and/or pharmaceutical modulation of myosin function. Aim 2: Develop growth and remodeling algorithms to predict chronic changes in ventricular structure and function resulting from genetic and/or pharmaceutical modulation of myosin function. Aim 3: Calibrate and validate the model using experimental data quantifying different spatial and temporal scales.

Abstract Familial cardiomyopathies cause long-term geometrical and functional changes in the heart. Hypertrophic disease is typically associated with larger myocytes, thicker ventricular walls, myocyte disarray, and fibrosis. It's not yet clear how the mutations that impact myosin function scale up to induce these multiscaled effects. The parent grant proposed to develop growth and remodeling laws that couple tissue-level deformation to long-term changes in molecular-level structure and function (via alterations in myosin kinetics, sarcomere number/size, and fibrosis). This will allow the model to evolve over time, either remodeling in response to deleterious mutations, or reverse remodeling (that is, undergoing beneficial changes) as a result of therapeutic interventions. One of the key phenotypes listed above, which is not explored in depth in the parent grant, is the development of myocyte disarray in the ventricular wall. This ?disorganization? of myofibers can lead to detrimental changes in the deformation patterns in the heart, which will ultimately degrade global pump function. It has been shown in patients with hypertrophic cardiomyopathy (HCM) that regional myofiber disarray is linked to ventricular hypokinesis. Despite this clinical relevance, few studies have tried to address the growth and remodeling laws that are needed to characterize myofiber reorientation in the presence of HCM. The research planned in this supplement seeks to overcome these limitations and develop a new growth and remodeling law that will relate changes in myocardial microstructure (via myofiber disarray) and ventricular strain patterns. This new law will enhance the ongoing work in the parent grant by coupling changes in ventricular wall volume (parent grant) to deformation driven changes in myofiber orientation (supplement). Together, this law will more accurately capture the structural alterations caused by HCM. To achieve this, the following aims are proposed for the supplement period: Aim 1: Quantify myofiber disarray with diffusion tensor (DT) MRI at the same time points as the strain patterns collected with DENSE MRI. It should be noted that the DENSE MRI data is being collected as part of the parent grant. The DT MRI data will be collected at multiple time points to capture the evolution of myofiber disarray. Aim 2: Implement a new growth and remodeling law into the finite element framework to account for myofiber disarry. The finite element code is organized in a modular way, which will allow for seamless integration of the new growth and remodeling law with what is being implemented for the parent grant.

PROJECT ABSTRACT In this project, we will develop a new computational modeling framework capable of designing targeted molecular therapies for heart failure. Impairment of cardiac muscle function constitutes a major clinical problem and comes in many forms. For example, sarcomere-level contraction is depressed in many of the 3 million Americans who have Heart Failure with reduced Ejection Fraction (HFrEF). The opposite issue, excessive activity of muscle proteins, can contribute to Heart Failure with preserved Ejection Fraction (HFpEF) by slowing relaxation and stiffening the ventricle. Genetic mutations to sarcomeric proteins afflict another 700,000 Americans. Gain of function mutations typically produce cardiac hypertrophy while loss of molecular function results in dilated cardiomyopathy. Patients and physicians urgently need better therapies for these conditions but the clinical trials used to test potential new strategies cost ~$1 billion and are plagued by high failure rates. This project tests the hypothesis that computer modeling can help to overcome these challenges by efficiently predicting the therapeutic potential of novel drug targets in the context of each different form of heart failure. The ultimate goal would be to screen a wide range of molecular strategies in silico and then select the most promising options for animal experiments and/or clinical trials. In the long term, it might even be possible to implement patient-specific computer modeling to help optimize treatment plans. The more immediate impacts would include reducing costs and focusing trials on the most effective molecular targets. The first step is to establish the feasibility of a modeling-driven pipeline using murine models of heart failure (HF) and a single molecular target. Recent studies show that sarcomere-focused treatments for HF have significant promise and that myosin-binding protein-C (MyBPC) could be a particularly effective target. This is because MyBPC can both enhance and inhibit contractility with the net regulatory effect depending on the phosphorylation status of three known residues. Phospho-variants of MyBPC could therefore be engineered to increase or decrease cardiac contractility as desired. In our view, the main roadblock hindering MyBPC's development as a potential new therapy is incomplete understanding of the molecule's mechanistic action. Specifically, it is not yet known precisely how the phosphorylation status of each residue modulates MyBPC's ability to enhance function (by activating the thin filament) and depress function (by restricting the mobility of detached myosin heads). The goals of this project are therefore to (1) develop a modeling framework that establishes how site-specific MyBPC phosphorylation impacts contractile function, (2) validate the model using sarcomere to animal-level experiments, and (3) test the pipeline's ability to predict effective therapeutic strategies by combining in silico screening and viral delivery of computer-selected mutant MyBPC.

Abstract The capacity of the ventricles to perform work (i.e., generate power) is essential for moving blood throughout the circulatory systems. Ventricular power is determined by the power generating capacity of the myofilaments within the cardiac myocyte. However, the sub-cellular processes that regulate myofilament power are incompletely understood. The overall objective of this proposal is to use biochemical, biophysical, and transgenic tools to discern (i) thin filament and (ii) thick filament-based mechanisms that regulate power and (iii) integrate these control mechanisms into a computational model that can predict how sarcomere-level modifications impact hemodynamics. The two mechanistic hypotheses are (Aim 1) alterations in the functional rigidity of thin filament regulatory units modulate cooperative recruitment of cross-bridges, which, in turn, determines power and (Aim 2) phosphorylation of myosin binding protein- C (MyBP-C) per se increases myofibrillar power output by three distinct biophysical mechanisms. In (Aim 3), a multi-state kinetic model of sarcomeric power output will be generated whereby thin and thick filament dynamic properties can be manipulated and evaluated for functional impacts to cooperativity and power. Aim 3 goes beyond the sarcomere and uses multiscale modeling to predict how strategic manipulation of myofilament targets will impact ventricular function and hemodynamics, which will be experimentally tested in a hypothesis-driven manner. Multi-scale modeling will provide a new platform to interrogate biophysical modifications that produce the largest functional effects and, thus, illuminate high-value therapeutic targets to optimize ventricular performance in patients with genetic and adaptive cardiomyopathies.

ABSTRACT This translational project uses human biospecimens procured from organ donors and patients undergoing cardiac transplant to advance understanding of a cellular-level mechanism that underpins the Frank-Starling relationship. Specifically, the project focuses on length-dependent activation, defined as the increased maximum force and Ca2+ sensitivity of contraction induced by myocardial stretch. The mechanisms that underlie length- dependent activation remain unclear, but may involve thick-filament regulation and transitions between the newly discovered OFF and ON states of myosin. Co-PI Campbell has spent a decade building a biobank that now contains >10,000 myocardial specimens from >360 patients. Pilot experiments performed by Co-PI Tanner with these samples show that length-dependent changes in Ca2+ sensitivity are eliminated in myocardium from patients who have non-ischemic heart failure, but preserved in myocardium from organ donors and patients who have ischemic heart failure. New computer modeling predicts that these functional effects reflect destabilization of the myosin OFF state in patients who have non-ischemic heart failure. This hypothesis is supported by additional experiments that used fluorescent polarization techniques to assess OFF/ON dynamics in the thick filaments of human myocardium. Further pilot studies tested the effects of peptides targeted to the thick filament. Peptides that stabilize the OFF state reduced the Ca2+ sensitivity of contraction at long sarcomere length while destabilizing peptides enhanced Ca2+ sensitivity at short length. The length-dependence of these effects was predicted by our computer modeling. The project builds on these data from human biospecimens and integrates the skills and resources of five cardiovascular researchers, a statistician, and a physician-scientist who specializes in advanced heart failure. The Aims explore the global hypothesis that length-dependent activation is reduced in patients who have non- ischemic heart failure because their cardiac thick filaments are biased towards the ON state. Aim 1: Test the hypothesis that length-dependent changes in Ca2+ sensitivity are reduced in myocardium from patients who have non-ischemic heart failure. Aim 2: Test the hypothesis that the OFF state of the thick filament is destabilized in myocardium from patients who have non-ischemic heart failure. Aim 3: Target OFF/ON transitions to manipulate the Ca2+ sensitivity of human myocardium.