Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure.

KEY POINTS: The present study investigated the mechanism associated with impaired cardiac mechanosensing that leads to heart failure by examining the factors regulating muscle LIM protein subcellular distribution in myocytes. In myocytes, muscle LIM protein subcellular distribution is regulated by cell contractility rather than passive stretch via heme oxygenase-1 and histone deacetylase signalling. The result of the present study provide new insights into mechanotransduction in cardiac myocytes. Myocyte mechanosensitivity, as indicated by the muscle LIM protein ratio, is also correlated with cardiac function in the transition to failure in a guinea-pig model of disease. This shows that the loss mechanosensitivity plays an important role during the transition to failure in the heart. The present study provides the first indication that mechanosensing could be modified pharmacologically during the transition to heart failure. ABSTRACT: Impaired mechanosensing leads to heart failure and a decreased ratio of cytoplasmic to nuclear CSRP3/muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. In the present study, we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to a 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased by 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 µm blebbistatin resulted in an ∼3-fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signalling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme oxygenase1 (HO-1) activity with protoporphyrin IX zinc(II) blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea-pig model of angiotensin II infusion (400 ng kg(-1)  min(-1) ) over 12 weeks. Using subcellular fractionation, we showed that the MLP ratio increased by 88% (n = 4, P < 0.01) during compensated hypertrophy but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01, n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signalling.

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte.

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

Depressed myocardial cross-bridge cycling kinetics in a female guinea pig model of diastolic heart failure.

Cardiac hypertrophy is associated with diastolic heart failure (DHF), a syndrome in which systolic function is preserved but cardiac filling dynamics are depressed. The molecular mechanisms underlying DHF and the potential role of altered cross-bridge cycling are poorly understood. Accordingly, chronic pressure overload was induced by surgically banding the thoracic ascending aorta (AOB) in ∼400 g female Dunkin Hartley guinea pigs (AOB); Sham-operated age-matched animals served as controls. Guinea pigs were chosen to avoid the confounding impacts of altered myosin heavy chain (MHC) isoform expression seen in other small rodent models. In vivo cardiac function was assessed by echocardiography; cardiac hypertrophy was confirmed by morphometric analysis. AOB resulted in left ventricle (LV) hypertrophy and compromised diastolic function with normal systolic function. Biochemical analysis revealed exclusive expression of ß-MHC isoform in both sham control and AOB LVs. Myofilament function was assessed in skinned multicellular preparations, skinned single myocyte fragments, and single myofibrils prepared from frozen (liquid N2) LVs. The rates of force-dependent ATP consumption (tension-cost) and force redevelopment (Ktr), as well as myofibril relaxation time (Timelin) were significantly blunted in AOB, indicating reduced cross-bridge cycling kinetics. Maximum Ca2+ activated force development was significantly reduced in AOB myocytes, while no change in myofilament Ca2+ sensitivity was observed. Our results indicate blunted cross-bridge cycle in a ß-MHC small animal DHF model. Reduced cross-bridge cycling kinetics may contribute, at least in part, to the development of DHF in larger mammals, including humans.

Direct 3D bioprinting of cardiac micro-tissues mimicking native myocardium.

The heart possesses a complex three-dimensional (3D) laminar myofiber organization; however, because engineering physiologically relevant 3D tissues remains a technical challenge, the effects of cardiomyocyte alignment on excitation-contraction coupling, shortening and force development have not been systematically studied. Cellular shape and orientations in 3D can be controlled by engineering scaffold microstructures and encapsulating cells near these geometric cues. Here, we show that a novel method of cell encapsulation in 3D methacrylated gelatin (GelMA) scaffolds patterned via Microscale Continuous Optical Printing (µCOP) can rapidly micropattern neonatal mouse ventricular cardiomyocytes (NMVCMs) in photocrosslinkable hydrogels. Encapsulated cardiomyocytes preferentially align with the engineered microarchitecture and can display morphology and myofibril alignment phenotypic of myocardium in vivo. Utilizing the µCOP system, an asymmetric, multi-material, cantilever-based scaffold was directly printed, so that the force produced by the microtissue was transmitted onto a single deformable pillar. Aligned 3D encapsulated NMVCM scaffolds produced nearly 2 times the force compared to aligned 2D seeded samples. To further highlight the flexibility of µCOP, NMVCMs were encapsulated in several patterns to compare the effects of varying degrees of alignment on tissue displacement and synchronicity. Well aligned myofiber cultured patterns generated 4-10 times the contractile force of less anisotropically patterned constructs. Finally, normalized fluo-4 fluorescence of NMVCM-encapsulated structures showed characteristic calcium transient waveforms that increased in magnitude and rate of decline during treatment with 100 nM isoproterenol. This novel instrumented 3D cardiac microtissue serves as a physiologically relevant in vitro model system with great potential for use in cardiac disease modeling and drug screening.

3D printed micro-scale force gauge arrays to improve human cardiac tissue maturation and enable high throughput drug testing.

Human induced pluripotent stem cell - derived cardiomyocytes (iPSC-CMs) are regarded as a promising cell source for establishing in-vitro personalized cardiac tissue models and developing therapeutics. However, analyzing cardiac force and drug response using mature human iPSC-CMs in a high-throughput format still remains a great challenge. Here we describe a rapid light-based 3D printing system for fabricating micro-scale force gauge arrays suitable for 24-well and 96-well plates that enable scalable tissue formation and measurement of cardiac force generation in human iPSC-CMs. We demonstrate consistent tissue band formation around the force gauge pillars with aligned sarcomeres. Among the different maturation treatment protocols we explored, 3D aligned cultures on force gauge arrays with in-culture pacing produced the highest expression of mature cardiac marker genes. We further demonstrated the utility of these micro-tissues to develop significantly increased contractile forces in response to treatment with isoproterenol, levosimendan, and omecamtiv mecarbil. Overall, this new 3D printing system allows for high flexibility in force gauge design and can be optimized to achieve miniaturization and promote cardiac tissue maturation with great potential for high-throughput in-vitro drug screening applications. STATEMENT OF SIGNIFICANCE: The application of iPSC-derived cardiac tissues in translatable drug screening is currently limited by the challenges in forming mature cardiac tissue and analyzing cardiac forces in a high-throughput format. We demonstrate the use of a rapid light-based 3D printing system to build a micro-scale force gauge array that enables scalable cardiac tissue formation from iPSC-CMs and measurement of contractile force development. With the capability to provide great flexibility over force gauge design as well as optimization to achieve miniaturization, our 3D printing system serves as a promising tool to build cardiac tissues for high-throughput in-vitro drug screening applications.

Insights and Challenges of Multi-Scale Modeling of Sarcomere Mechanics in cTn and Tm DCM Mutants-Genotype to Cellular Phenotype.

Dilated Cardiomyopathy (DCM) is a leading cause of sudden cardiac death characterized by impaired pump function and dilatation of cardiac ventricles. In this review we discuss various in silico approaches to elucidating the mechanisms of genetic mutations leading to DCM. The approaches covered in this review focus on bridging the spatial and temporal gaps that exist between molecular and cellular processes. Mutations in sarcomeric regulatory thin filament proteins such as the troponin complex (cTn) and Tropomyosin (Tm) have been associated with DCM. Despite the experimentally-observed myofilament measures of contractility in the case of these mutations, the mechanisms by which the underlying molecular changes and protein interactions scale up to organ failure by these mutations remains elusive. The review highlights multi-scale modeling approaches and their applicability to study the effects of sarcomeric gene mutations in-silico. We discuss some of the insights that can be gained from computational models of cardiac biomechanics when scaling from molecular states to cellular level.

Model of Human Fetal Growth in Hypoplastic Left Heart Syndrome: Reduced Ventricular Growth Due to Decreased Ventricular Filling and Altered Shape.

INTRODUCTION: Hypoplastic left heart syndrome (HLHS) is a congenital condition with an underdeveloped left ventricle (LV) that provides inadequate systemic blood flow postnatally. The development of HLHS is postulated to be due to altered biomechanical stimuli during gestation. Predicting LV size at birth using mid-gestation fetal echocardiography is a clinical challenge critical to prognostic counseling. HYPOTHESIS: We hypothesized that decreased ventricular filling in utero due to mitral stenosis may reduce LV growth in the fetal heart via mechanical growth signaling. METHODS: We developed a novel finite element model of the human fetal heart in which cardiac myocyte growth rates are a function of fiber and cross-fiber strains, which is affected by altered ventricular filling, to simulate alterations in LV growth and remodeling. Model results were tested with echocardiogram measurements from normal and HLHS fetal hearts. RESULTS: A strain-based fetal growth model with a normal 22-week ventricular filling (1.04 mL) was able to replicate published measurements of changes between mid-gestation to birth of mean LV end-diastolic volume (EDV) (1.1-8.3 mL) and dimensions (long-axis, 18-35 mm; short-axis, 9-18 mm) within 15% root mean squared deviation error. By decreasing volumetric load (-25%) at mid-gestation in the model, which emulates mitral stenosis in utero, a 65% reduction in LV EDV and a 46% reduction in LV wall volume were predicted at birth, similar to observations in HLHS patients. In retrospective blinded case studies for HLHS, using mid-gestation echocardiographic data, the model predicted a borderline and severe hypoplastic LV, consistent with the patients' late-gestation data in both cases. Notably, the model prediction was validated by testing for changes in LV shape in the model against clinical data for each HLHS case study. CONCLUSION: Reduced ventricular filling and altered shape may lead to reduced LV growth and a hypoplastic phenotype by reducing myocardial strains that serve as a myocyte growth stimulus. The human fetal growth model presented here may lead to a clinical tool that can help predict LV size and shape at birth based on mid-gestation LV echocardiographic measurements.

Molecular Effects of cTnC DCM Mutations on Calcium Sensitivity and Myofilament Activation-An Integrated Multiscale Modeling Study.

Mutations in cardiac troponin C (D75Y, E59D, and G159D), a key regulatory protein of myofilament contraction, have been associated with dilated cardiomyopathy (DCM). Despite reports of altered myofilament function in these mutants, the underlying molecular alterations caused by these mutations remain elusive. Here we investigate in silico the intramolecular mechanisms by which these mutations affect myofilament contraction. On the basis of the location of cardiac troponin C (cTnC) mutations, we tested the hypothesis that intramolecular effects can explain the altered myofilament calcium sensitivity of force development for D75Y and E59D cTnC, whereas altered cardiac troponin C-troponin I (cTnC-cTnI) interaction contributes to the reported contractile effects of the G159D mutation. We employed a multiscale approach combining molecular dynamics (MD) and Brownian dynamics (BD) simulations to estimate cTnC calcium association and hydrophobic patch opening. We then integrated these parameters into a Markov model of myofilament activation to compute the steady-state force-pCa relationship. The analysis showed that myofilament calcium sensitivity with D75Y and E59D can be explained by changes in calcium binding affinity of cTnC and the rate of hydrophobic patch opening, if a partial cTnC interhelical opening angle (110°) is sufficient for cTnI switch peptide association to cTnC. In contrast, interactions between cTnC and cTnI within the cardiac troponin complex must also be accounted for to explain contractile alterations due to G159D. In conclusion, this is the first multiscale in silico study to elucidate how direct molecular effects of genetic mutations in cTnC translate to altered myofilament contractile function.

3D cardiac µtissues within a microfluidic device with real-time contractile stress readout.

We present the development of three-dimensional (3D) cardiac microtissues within a microfluidic device with the ability to quantify real-time contractile stress measurements in situ. Using a 3D patterning technology that allows for the precise spatial distribution of cells within the device, we created an array of 3D cardiac microtissues from neonatal mouse cardiomyocytes. We integrated the 3D micropatterning technology with microfluidics to achieve perfused cell-laden structures. The cells were encapsulated within a degradable gelatin methacrylate hydrogel, which was sandwiched between two polyacrylamide hydrogels. The polyacrylamide hydrogels were used as "stress sensors" to acquire the contractile stresses generated by the beating cardiac cells. The cardiac-specific response of the engineered 3D system was examined by exposing it to epinephrine, an adrenergic neurotransmitter known to increase the magnitude and frequency of cardiac contractions. In response to exogenous epinephrine the engineered cardiac tissues exhibited an increased beating frequency and stress magnitude. Such cost-effective and easy-to-adapt 3D cardiac systems with real-time functional readout could be an attractive technological platform for drug discovery and development.