Machine learning-guided engineering of genetically encoded fluorescent calcium indicators.

Here we used machine learning to engineer genetically encoded fluorescent indicators, protein-based sensors critical for real-time monitoring of biological activity. We used machine learning to predict the outcomes of sensor mutagenesis by analyzing established libraries that link sensor sequences to functions. Using the GCaMP calcium indicator as a scaffold, we developed an ensemble of three regression models trained on experimentally derived GCaMP mutation libraries. The trained ensemble performed an in silico functional screen on 1,423 novel, uncharacterized GCaMP variants. As a result, we identified the ensemble-derived GCaMP (eGCaMP) variants, eGCaMP and eGCaMP+, which achieve both faster kinetics and larger ∆F/F0 responses upon stimulation than previously published fast variants. Furthermore, we identified a combinatorial mutation with extraordinary dynamic range, eGCaMP2+, which outperforms the tested sixth-, seventh- and eighth-generation GCaMPs. These findings demonstrate the value of machine learning as a tool to facilitate the efficient engineering of proteins for desired biophysical characteristics.

The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness.

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT 90 ) and time to 90% Ca 2+ transient decay (DT 90 ), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT 90 and partially normalized DT 90 , suggesting improved Ca 2+ handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca 2+ transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.

Danicamtiv Increases Myosin Recruitment and Alters Cross-Bridge Cycling in Cardiac Muscle.

BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficit. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the ON state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ. CONCLUSIONS: As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.

Cell based dATP delivery as a therapy for chronic heart failure.

Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular performance when delivered acutely post-myocardial infarction but are ineffective in chronic myocardial infarction/heart failure. 2'-deoxy-ATP (dATP) activates cardiac myosin and potently increases contractility. Here we engineered hPSC-CMs to overexpress ribonucleotide reductase, the enzyme controlling dATP production. In vivo, dATP-producing CMs formed new myocardium that transferred dATP to host cardiomyocytes via gap junctions, increasing their dATP levels. Strikingly, when transplanted into chronically infarcted hearts, dATP-producing grafts increased left ventricular function, whereas heart failure worsened with wild-type grafts or vehicle injections. dATP-donor cells recipients had greater voluntary exercise, improved cardiac metabolism, reduced pulmonary congestion and pathological cardiac hypertrophy, and improved survival. This combination of remuscularization plus enhanced host contractility offers a novel approach to treating the chronically failing heart.

Machine learning meets Monte Carlo methods for models of muscle's molecular machinery to classify mutations.

The timing and magnitude of force generation by a muscle depend on complex interactions in a compliant, contractile filament lattice. Perturbations in these interactions can result in cardiac muscle diseases. In this study, we address the fundamental challenge of connecting the temporal features of cardiac twitches to underlying rate constants and their perturbations associated with genetic cardiomyopathies. Current state-of-the-art metrics for characterizing the mechanical consequence of cardiac muscle disease do not utilize information embedded in the complete time course of twitch force. We pair dimension reduction techniques and machine learning methods to classify underlying perturbations that shape the timing of twitch force. To do this, we created a large twitch dataset using a spatially explicit Monte Carlo model of muscle contraction. Uniquely, we modified the rate constants of this model in line with mouse models of cardiac muscle disease and varied mutation penetrance. Ultimately, the results of this study show that machine learning models combined with biologically informed dimension reduction techniques can yield excellent classification accuracy of underlying muscle perturbations.

Danicamtiv increases myosin recruitment and alters the chemomechanical cross bridge cycle in cardiac muscle.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the "on" state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danicamtiv corrected calcium sensitivity in demembranated and abnormal twitch magnitude and kinetics in intact cardiac tissue. Significance Statement: Directly augmenting sarcomere function has potential to overcome limitations of currently used inotropic agents to improve cardiac contractility. Myosin modulation is a novel mechanism for increased contraction in cardiomyopathies. Danicamtiv is a myosin activator that is currently under investigation for use in cardiomyopathy patients. Our study is the first detailed mechanism of how Danicamtiv increases force and alters kinetics of cardiac activation and relaxation. This new understanding of the mechanism of action of Danicamtiv can be used to help identify patients that could benefit most from this treatment.

Correcting dilated cardiomyopathy with fibroblast-targeted p38 deficiency.

Inherited mutations in contractile and structural genes, which decrease cardiomyocyte tension generation, are principal drivers of dilated cardiomyopathy (DCM)- the leading cause of heart failure 1,2 . Progress towards developing precision therapeutics for and defining the underlying determinants of DCM has been cardiomyocyte centric with negligible attention directed towards fibroblasts despite their role in regulating the best predictor of DCM severity, cardiac fibrosis 3,4 . Given that failure to reverse fibrosis is a major limitation of both standard of care and first in class precision therapeutics for DCM, this study examined whether cardiac fibroblast-mediated regulation of the heart's material properties is essential for the DCM phenotype. Here we report in a mouse model of inherited DCM that prior to the onset of fibrosis and dilated myocardial remodeling both the myocardium and extracellular matrix (ECM) stiffen from switches in titin isoform expression, enhanced collagen fiber alignment, and expansion of the cardiac fibroblast population, which we blocked by genetically suppressing p38α in cardiac fibroblasts. This fibroblast-targeted intervention unexpectedly improved the primary cardiomyocyte defect in contractile function and reversed ECM and dilated myocardial remodeling. Together these findings challenge the long-standing paradigm that ECM remodeling is a secondary complication to inherited defects in cardiomyocyte contractile function and instead demonstrate cardiac fibroblasts are essential contributors to the DCM phenotype, thus suggesting DCM-specific therapeutics will require fibroblast-specific strategies.

dATP elevation induces myocardial metabolic remodeling to support improved cardiac function.

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ∼1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs.

Modulating the tension-time integral of the cardiac twitch prevents dilated cardiomyopathy in murine hearts.

Dilated cardiomyopathy (DCM) is often associated with sarcomere protein mutations that confer reduced myofilament tension-generating capacity. We demonstrated that cardiac twitch tension-time integrals can be targeted and tuned to prevent DCM remodeling in hearts with contractile dysfunction. We employed a transgenic murine model of DCM caused by the D230N-tropomyosin (Tm) mutation and designed a sarcomere-based intervention specifically targeting the twitch tension-time integral of D230N-Tm hearts using multiscale computational models of intramolecular and intermolecular interactions in the thin filament and cell-level contractile simulations. Our models predicted that increasing the calcium sensitivity of thin filament activation using the cardiac troponin C (cTnC) variant L48Q can sufficiently augment twitch tension-time integrals of D230N-Tm hearts. Indeed, cardiac muscle isolated from double-transgenic hearts expressing D230N-Tm and L48Q cTnC had increased calcium sensitivity of tension development and increased twitch tension-time integrals compared with preparations from hearts with D230N-Tm alone. Longitudinal echocardiographic measurements revealed that DTG hearts retained normal cardiac morphology and function, whereas D230N-Tm hearts developed progressive DCM. We present a computational and experimental framework for targeting molecular mechanisms governing the twitch tension of cardiomyopathic hearts to counteract putative mechanical drivers of adverse remodeling and open possibilities for tension-based treatments of genetic cardiomyopathies.

Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2'-deoxy-ATP.

KEY POINTS: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation. The naturally occurring nucleotide 2-deoxy-ATP (dATP) is a myosin activator that enhances cross-bridge binding and kinetics. X-ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin-binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation. This study of the dATP-induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin-targeted molecular compounds to treat muscle diseases. ABSTRACT: Here we used time-resolved small-angle X-ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first-order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6 ) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2-deoxy-ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X-ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin-binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions.

Rapamycin persistently improves cardiac function in aged, male and female mice, even following cessation of treatment.

Even in healthy aging, cardiac morbidity and mortality increase with age in both mice and humans. These effects include a decline in diastolic function, left ventricular hypertrophy, metabolic substrate shifts, and alterations in the cardiac proteome. Previous work from our laboratory indicated that short-term (10-week) treatment with rapamycin, an mTORC1 inhibitor, improved measures of these age-related changes. In this report, we demonstrate that the rapamycin-dependent improvement of diastolic function is highly persistent, while decreases in both cardiac hypertrophy and passive stiffness are substantially persistent 8 weeks after cessation of an 8-week treatment of rapamycin in both male and female 22- to 24-month-old C57BL/6NIA mice. The proteomic and metabolomic abundance changes that occur after 8 weeks of rapamycin treatment have varying persistence after 8 further weeks without the drug. However, rapamycin did lead to a persistent increase in abundance of electron transport chain (ETC) complex components, most of which belonged to Complex I. Although ETC protein abundance and Complex I activity were each differentially affected in males and females, the ratio of Complex I activity to Complex I protein abundance was equally and persistently reduced after rapamycin treatment in both sexes. Thus, rapamycin treatment in the aged mice persistently improved diastolic function and myocardial stiffness, persistently altered the cardiac proteome in the absence of persistent metabolic changes, and led to persistent alterations in mitochondrial respiratory chain activity. These observations suggest that an optimal translational regimen for rapamycin therapy that promotes enhancement of healthspan may involve intermittent short-term treatments.

Cardiac myosin activation with 2-deoxy-ATP via increased electrostatic interactions with actin.

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

Gene Therapy Rescues Cardiac Dysfunction in Duchenne Muscular Dystrophy Mice by Elevating Cardiomyocyte Deoxy-Adenosine Triphosphate.

Mutations in the gene encoding for dystrophin leads to structural and functional deterioration of cardiomyocytes and is a hallmark of cardiomyopathy in Duchenne muscular dystrophy (DMD) patients. Administration of recombinant adeno-associated viral vectors delivering microdystrophin or ribonucleotide reductase (RNR), under muscle-specific regulatory control, rescues both baseline and high workload-challenged hearts in an aged, DMD mouse model. However, only RNR treatments improved both systolic and diastolic function under those conditions. Cardiac-specific recombinant adeno-associated viral treatment of RNR holds therapeutic promise for improvement of cardiomyopathy in DMD patients.

Fast and sensitive HPLC-MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells.

Deoxyribonucleoside triphosphates (dNTPs) are used in DNA synthesis and repair. Even slight imbalances can have adverse biological effects. This study validates a fast and sensitive HPLC-MS/MS method for direct quantification of intracellular dNTPs from tissue. Equal volumes of methanol and water were used for nucleotide extraction from mouse heart and gastrocnemius muscle and isolated cardiomyocytes followed by centrifugation to remove particulates. The resulting supernatant was analyzed on a porous graphitic carbon chromatography column using an elution gradient of ammonium acetate in water and ammonium hydroxide in acetonitrile with a run time of just 10min. Calibration curves of all dNTPs ranged from 62.5 to 2500fmol injections and demonstrated excellent linearity (r2>0.99). The within day and between day precision, as measured by the coefficient of variation (CV (%)), was <25% for all points, including the lower limit of quantification (LLOQ). The inter-day accuracy was within 12% of expected concentration for the LLOQ and within 7% for all other points on the calibration curve. The intra-day accuracy was within 22% for the LLOQ and within 11% for all points on the curve. Compared to existing methods, this study presents a faster and more sensitive method for dNTP quantification.

Heart failure with preserved ejection fraction and skeletal muscle physiology.

Heart failure with preserved ejection fraction (HFpEF) accounts for half of all heart failure in the USA, increases in prevalence with aging, and has no effective therapies. Intriguingly, the pathophysiology of HFpEF has many commonalities with the aged cardiovascular system including reductions in diastolic compliance, chronotropic defects, increased resistance in the peripheral vasculature, and poor energy substrate utilization. Decreased exercise capacity is a cardinal symptom of HFpEF. However, its severity is often out of proportion to changes in cardiac output. This observation has led to studies of muscle function in HFpEF revealing structural, biomechanical, and metabolic changes. These data, while incomplete, support a hypothesis that similar to aging, HFPEF is a systemic process. Understanding the mechanisms leading to exercise intolerance in this condition may lead to strategies to improve morbidity in both HFpEF and aging.

A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy.

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Translation of Cardiac Myosin Activation with 2-deoxy-ATP to Treat Heart Failure via an Experimental Ribonucleotide Reductase-Based Gene Therapy.

Despite recent advances, chronic heart failure remains a significant and growing unmet medical need, reaching epidemic proportions carrying substantial morbidity, mortality, and costs. A safe and convenient therapeutic agent that produces sustained inotropic effects could ameliorate symptoms, and improve functional capacity and quality of life. We discovered small amounts of 2-deoxy-ATP (dATP) activate cardiac myosin leading to enhanced contractility in normal and failing heart muscle. Cardiac myosin activation triggers faster myosin crossbridge cycling with greater force generation during each contraction. We describe the rationale and results of a translational medicine effort to increase dATP levels using a gene therapy strategy that upregulates ribonucleotide reductase, the rate-limiting enzyme for dATP synthesis, selectively in cardiomyocytes. In small and large animal models of heart failure, a single dose of this gene therapy has led to sustained inotropic effects with no toxicity or safety concerns identified to-date. Further animal studies are being conducted with the goal of testing this agent in patients with heart failure.

AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility.

Impaired systolic function, resulting from acute injury or congenital defects, leads to cardiac complications and heart failure. Current therapies slow disease progression but do not rescue cardiac function. We previously reported that elevating the cellular 2 deoxy-ATP (dATP) pool in transgenic mice via increased expression of ribonucleotide reductase (RNR), the enzyme that catalyzes deoxy-nucleotide production, increases myosin-actin interaction and enhances cardiac muscle contractility. For the current studies, we initially injected wild-type mice retro-orbitally with a mixture of adeno-associated virus serotype-6 (rAAV6) containing a miniaturized cardiac-specific regulatory cassette (cTnT(455)) composed of enhancer and promotor portions of the human cardiac troponin T gene (TNNT2) ligated to rat cDNAs encoding either the Rrm1 or Rrm2 subunit. Subsequent studies optimized the system by creating a tandem human RRM1-RRM2 cDNA with a P2A self-cleaving peptide site between the subunits. Both rat and human Rrm1/Rrm2 cDNAs resulted in RNR enzyme overexpression exclusively in the heart and led to a significant elevation of left ventricular (LV) function in normal mice and infarcted rats, measured by echocardiography or isolated heart perfusions, without adverse cardiac remodeling. Our study suggests that increasing RNR levels via rAAV-mediated cardiac-specific expression provide a novel gene therapy approach to potentially enhance cardiac systolic function in animal models and patients with heart failure.