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We have used spectroscopic and functional assays to evaluate the effects of a group of actin-binding compounds on striated muscle protein structure and function. Actin is present in every human cell, and its interaction with multiple myosin isoforms and multiple actin-binding proteins is essential for cellular viability. A previous high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay from our group identified a class of compounds that bind to actin and affect actomyosin structure and function. In the current study, we tested their effects on the two isoforms of striated muscle -actins, skeletal and cardiac. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that these effects were different for the two actin isoforms, suggesting a different mode of action. To determine the effects of these compounds on sarcomeric function, we further tested their activity on skeletal and cardiac myofibrils. We found that several compounds affected ATPase activity of skeletal and cardiac myofibrils differently, suggesting different mechanisms of action of these compounds for the two muscle types. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

Muscle myosins use the energy from ATP hydrolysis to perform mechanical work during muscle contraction. Recent work suggests that the interactions between the catalytic and light-chain-binding domains of myosin, the S2 coiled-coil fragment, and the thick-filament backbone, stabilize an auto-inhibited structural state of myosin, termed the super-relaxed state (SRX), with slowed ATP turnover kinetics. The super-relaxed state is hypothesized to play an important role in regulating contractility and thermogenesis in skeletal, cardiac muscle, and smooth muscle. We are using quantitative confocal microscopy of fluorescent ATP turnover in skinned skeletal and cardiac muscle fibers to measure the mole fraction of myosin molecules in the super-relaxed state, over a range of perturbations including animal age, sex, and hormonal state, post-translational modification of sarcomeric regulatory proteins, and small molecule modulators. From this work, we are mapping the determinants and regulatory correlates that control partitioning of myosin molecules into this important, poorly understood biophysical state. This work is supported by grants NIH R01AR032961-33 and T32AR007612-15 (to DDT).

Myosin's lever arm undergoes structural changes that are coupled to its biochemical state during ATPase cycling in muscle. Structural states of myosin's lever can be detected directly using the high-precision, high-throughput technique of transient time-resolved FRET, (TR)2FRET, revealing kinetics, thermodynamics, and allosteric coupling. We have previously used this technique to investigate mechanochemical coupling in skeletal myosin and cardiac myosin perturbed by the heart failure drug omecamtiv mecarbil. Here we investigate other small molecule modulators of cardiac myosins mechanochemistry: arachidonic acid, EMD 57003, and compounds developed by MyoKardia, under investigation for treating hypertrophic cardiomyopathy. We find that one Myk compound dramatically decreases the amplitude of the fast phase of the power stroke, but does not significantly affect the rate of this transition. These data suggest that the compound shifts the equilibrium constant for hydrolysis toward the pre-hydrolysis state. This is in sharp contrast to the effect of omecamtiv mecarbil (Cytokinetics) under investigation for systolic heart failure, which shifts the equilibrium constant toward the post-hydrolysis states. This work was supported by NIH AR032961 & AR057220 (DDT), American Heart Association Scientist Development Grant (JMM), and Graduate Excellence Fellowship-University of MN (JAR).

Introduction: Mitochondrial dysfunction is a hallmark of heart failure (HF), resulting in decreased energy production and impaired mitochondrial quality control (mitophagy) with resultant contractile dysfunction and cell death. The 18-kDa mitochondrial translocator protein (TSPO) has been considered a part of the mitochondrial permeability transition pore (mPTP) for a long time; however, its role in mPTP regulation is now debatable and the precise role of the TSPO in cardiac physiology and HF remains poorly understood.Objective: Determine the role of TSPO in a murine pressure-overload model of HF.Methods and Results: Conditional, cardiac-specific TSPO knockout (KO) mice were generated using the Cre-loxP system. Pressure overload by transverse aortic constriction (TAC) for 8 weeks significantly increased TSPO expression in wild type (WT) mice, but not in KO mice. While WT TAC mice showed a marked reduction in systolic function, KO TAC mice did not have a significant reduction in ejection fraction, and also exhibited fewer clinical HF signs (less cardiac dilation and fibrosis). TAC reduced mitochondrial calcium uptake and enhanced mPTP opening in permeabilized myocytes from WT animals. Calcium uptake was restored in TAC KO mice without significant effect on mPTP activity. The restoration of the mitochondrial calcium uptake coincided with reduced oxidative stress, improved oxygen consumption rate, and enhanced ATP generation and mitochondrial reserve capacity. In WT TAC, elevated mitochondrial accumulation of mCherry-Parkin1 was not coupled to the increase in LC3-autophagosomes formation indicating impaired mitophagy. In contrast, mitophagy was normalized in TSPO KO.Conclusions: These data suggest a novel mechanism to prevent HF at the cellular level via normalization of TSPO levels which leads to preservation of mitochondrial calcium uptake, oxidative phosphorylation, and mitochondrial mt-QC resulting in preservation of mitochondrial integrity and cell survival.

Islet amyloid polypeptide (IAPP), a hormone co-secreted with insulin by pancreatic β-cells, forms amyloids when overexpressed. IAPP amyloids accumulate in pancreatic islets and are a hallmark of type-2 diabetes mellitus (T2DM). Recently, IAPP amyloids were found in kidneys of T2DM patients, suggesting that IAPP can deposit in organs other than pancreas. Apparently, the toxic effects to cells are mediated by the preamyloid oligomers. Here, we investigated whether IAPP preamyloid oligomers are present in the heart in T2DM. Using immunochemistry, we found significantly increased levels of IAPP oligomers (by 18±3%) in failing hearts from T2DM humans vs. non-diabetics and in hearts from T2DM rats transgenic for human IAPP (by 88±15%) vs. T2DM rats expressing only rat non-amyloidogenic IAPP. Most likely, IAPP accumulation in the heart occurs in the pre-diabetic state, when IAPP is oversecreted in the blood. To investigate the acute effect of IAPP oligomers on cardiac function, we incubated isolated rat cardiac myocytes with exogenous IAPP (5 and 50 μM) for 1-2 h. 50 μM IAPP significantly increased Ca transient amplitude (by 73±19%) in myocytes contracting at 2 Hz. For rapid screening and analysis of sites susceptible for IAPP accumulation we designed a noninvasive water proton NMR protocol. We deciphered the magnetic signal of water surrounding preamyloid oligomers. The variation of this signal was correlated with population distributions of oligomers and fibrils by using immunochemistry and electron microscopy. We found that embryonic amyloids generate hyper-intense magnetic signals, which are distinct from hypo-intense magnetic signals induced by amyloid plaques. These results suggest that IAPP preamyloid oligomers contribute to cardiac dysfunction in T2DM. Water proton NMR may prove a useful non-invasive method for detecting these molecular entities in the heart.