Jeffrey H. Omens

The Cardiac Mechanical Stretch Sensor Machinery Involves a Z Disc Complex that Is Defective in a Subset of Human Dilated Cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.

Substrate Stiffness Affects the Functional Maturation of Neonatal Rat Ventricular Myocytes

Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. To investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10 kPa substrates developed aligned sarcomeres, whereas cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10 kPa, than on stiffer or softer substrates. Cardiomyocytes on 10 kPa gels also had the largest calcium transients, sarcoplasmic calcium stores, and sarcoplasmic/endoplasmic reticular calcium ATPase2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocyte maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances neonatal rat ventricular myocytes maturation. Deviations from ideal stiffness result in lower expression of sarcoplasmic/endoplasmic reticular calcium ATPase, less stored calcium, smaller calcium transients, and lower force. On very stiff substrates, this adaptation seems to involve RhoA kinase.

An FHL1-containing complex within the cardiomyocyte sarcomere mediates hypertrophic biomechanical stress responses in mice

The response of cardiomyocytes to biomechanical stress can determine the pathophysiology of hypertrophic cardiac disease, and targeting the pathways regulating these responses is a therapeutic goal. However, little is known about how biomechanical stress is sensed by the cardiomyocyte sarcomere to transduce intracellular hypertrophic signals or how the dysfunction of these pathways may lead to disease. Here, we found that four-and-a-half LIM domains 1 (FHL1) is part of a complex within the cardiomyocyte sarcomere that senses the biomechanical stress-induced responses important for cardiac hypertrophy. Mice lacking Fhl1 displayed a blunted hypertrophic response and a beneficial functional response to pressure overload induced by transverse aortic constriction. A link to the Galphaq (Gq) signaling pathway was also observed, as Fhl1 deficiency prevented the cardiomyopathy observed in Gq transgenic mice. Mechanistic studies demonstrated that FHL1 plays an important role in the mechanism of pathological hypertrophy by sensing biomechanical stress responses via the N2B stretch sensor domain of titin and initiating changes in the titin- and MAPK-mediated responses important for sarcomere extensibility and intracellular signaling. These studies shed light on the physiological regulation of the sarcomere in response to hypertrophic stress.

Residual strain in rat left ventricle.

Residual stress in an organ is defined as the stress that remains when all external loads are removed. Residual stress has generally been ignored in published papers on left ventricular wall stress. To take residual stress into account in the analysis of stress distributions in a beating heart, one must first measure the residual strain in the no-load state of the heart. Residual strains in equatorial cross-sectional rings (2-3 mm thick) of five potassium-arrested rat left ventricles were measured. The effects of friction and external loading were reduced by submersing the specimen in fluid, and a hypothermic, hyperkalemic arresting solution containing nifedipine and EGTA was used to delay the onset of ischemic contracture. Stainless steel microspheres (60-100 microns) were lightly imbedded on the surface of the slices, and the coordinates of the microspheres were digitized from photographs taken before and after a radial cut was made through the left ventricular free wall. Two-dimensional strains computed from the deformation of a slice after one radial cut were defined as the residual strains in that slice. It was found that the distributions of the principal residual stretch ratios were asymmetric with respect to the radial cut: in areas where substantial transmural strain gradients existed, the distributions of strain components were different on the two sides of the radial cut. A second radial cut produced deformations significantly smaller than those produced from the first radial cut. Hence, a slice with one radial cut may be considered stress free.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection against myocardial dysfunction after a brief ischemic period in transgenic mice expressing inducible heat shock protein 70.

Brief ischemic periods lead to myocardial dysfunction without myocardial infarction. It has been shown that expression of inducible HSP70 in hearts of transgenic mice leads to decreased infarct size, but it remains unclear if HSP70 can also protect against myocardial dysfunction after brief ischemia. To investigate this question, we developed a mouse model in which regional myocardial function can be measured before and after a temporary ischemic event in vivo. In addition, myocardial function was determined after brief episodes of global ischemia in an isolated Langendorff heart. HSP70-positive mice and transgene negative littermates underwent 8 min of regional myocardial ischemia created by occlusion of the left descending coronary artery, followed by 60 min of reperfusion. This procedure did not result in a myocardial infarction. Regional epicardial strain was used as a sensitive indicator for changes in myocardial function after cardiac ischemia. Maximum principal strain was significantly greater in HSP70-positive mice with 88+/-6% of preischemic values vs. 58+/-6% in transgene-negative mice (P < 0.05). Similarly, in isolated Langendorff perfused hearts of HSP70-positive and transgene-negative littermates exposed to 10 min of global ischemia and 90 min of reperfusion, HSP70 transgenic hearts showed a better-preserved ventricular peak systolic pressure. Thus, we conclude that expression of HSP70 protects against postischemic myocardial dysfunction as shown by better preserved myocardial function.

Automated measurement of myofiber disarray in transgenic mice with ventricular expression of ras

Quantitative assessment of myofiber disarray associated with diseases such as familial hypertrophic cardiomyopathy (FHC) can be performed by estimating local angular deviation of fiber orientation in histologic sections. The large number of measurements required to estimate angular deviation prohibits manual measurement. We describe methods for automated measurement of local orientation and angular deviation in tissue sections from transgenic mice with ventricular expression of ras, proposed as a model of FHC.

Early Short-Term Treatment With Doxycycline Modulates Postinfarction Left Ventricular Remodeling

Background—Myocardial infarction (MI) is associated with early metalloproteinase (MMP) activation and extracellular matrix (ECM) degradation. We hypothesized that preserving the original ECM of the infarcted left ventricle (LV) by use of early short-term doxycycline (DOX) treatment preserves cardiac structure and function. Methods and Results—LV morphometry and function were measured in 3 groups of rats (sham, MI, and MI+DOX). DOX (30 mg/kg per day) was given orally 48 hours before and 48 hours after MI. Rats were examined at 2 and 4 weeks after MI. By 4 weeks, DOX significantly decreased (P <0.05 versus MI) the heart weight to body weight ratio, myocyte cross-sectional area, and internal LV diameter, whereas it preserved anterior wall thickness within the infarct. Collagen/muscle area fraction did not change in the region of the infarct/scar. Parallel left shifts (versus MI) were observed in pressure-volume relationships of DOX MI rats at all pressures. DOX treatment also shifted passive epicardial strains within the scar area toward normal values. No differences were observed in LV end-diastolic or peak systolic pressures, peak positive or negative LV dP/dt, or isovolumic relaxation rates. Assessment of LV global MMP and MMP-2/9 activities 1 hour after MI using fluorescent probes yielded significant differences with DOX. Conclusions—Brief, early MMP inhibition after MI yields preservation of LV structure and global as well as scar area passive function, supporting the concept that preserving the original ECM early after coronary occlusion lessens ventricular remodeling.

Myocardial Mechanics and Collagen Structure in the Osteogenesis Imperfecta Murine (oim)

Because the amount and structure of type I collagen are thought to affect the mechanics of ventricular myocardium, we investigated myocardial collagen structure and passive mechanical function in the osteogenesis imperfecta murine (oim) model of pro-&agr;2(I) collagen deficiency, previously shown to have less collagen and impaired biomechanics in tendon and bone. Compared with wild-type littermates, homozygous oim hearts exhibited 35% lower collagen area fraction (P <0.05), 38% lower collagen fiber number density (P <0.05), and 42% smaller collagen fiber diameter (P <0.05). Compared with wild-type, oim left ventricular (LV) collagen concentration was 45% lower (P <0.0001) and nonreducible pyridinoline cross-link concentration was 22% higher (P <0.03). Mean LV volume during passive inflation from 0 to 30 mm Hg in isolated hearts was 1.4-fold larger for oim than wild-type (P =NS). Uniaxial stress-strain relations in resting right ventricular papillary muscles exhibited 60% greater strains (P <0.01), 90% higher compliance (P =0.05), and 64% higher nonlinearity (P <0.05) in oim. Mean opening angle, after relief of residual stresses in resting LV myocardium, was 121±9 degrees in oim compared with 45±4 degrees in wild-type (P <0.0001). Mean myofiber angle in oim was 23±8 degrees greater than wild-type (P <0.02). Decreased myocardial collagen diameter and amount in oim is associated with significantly decreased fiber and chamber stiffness despite modestly increased collagen cross-linking. Altered myofiber angles and residual stress may be beneficial adaptations to these mechanical alterations to maintain uniformity of transmural fiber strain. In addition to supporting and organizing myocytes, myocardial collagen contributes directly to ventricular stiffness at high and low loads and can influence stress-free state and myofiber architecture.

Measurement of strain and analysis of stress in resting rat left ventricular myocardium

A technique has been developed for measuring two-dimensional strains in the left ventricle of the isolated arrested rat heart subjected to passive ventricular loading. The pressure-volume relationship was found in eight hearts during inflation of a left ventricular balloon. With the zero-pressure state as reference, in-plane strain components were determined using a triangle of ultrasonic dimension transducers (0.6-0.8 mm diameter) placed 3-6 mm apart in the midwall of the left ventricle. Mean circumferential (fiber) strain was larger than longitudinal (cross-fiber) strain (0.108 +/- 0.045, 0.055 +/- 0.045, respectively, at 11 mmHg), and shear strain (-0.048 +/- 0.029) was negative, consistent with left-handed torsion. The in-plane angle of greatest stretch was uniform with inflation (range = -26.5 degrees to -34.5 degrees). The equatorial region of the left ventricle was modeled with finite element analysis of a transversely isotropic thick-walled cylindrical shell subjected to internal loading and axial forces. The material parameters of an exponential strain energy function were optimized so that the least-squares difference between the predicted and the measured midwall strains was minimized. Material properties, stress and strain in the rat heart were compared to values predicted for the dog. In both species the tissue was stiffer in the fiber direction than in the cross-fiber direction. The ratio of fiber to cross-fiber stiffness was lower in the rat (2.50) than in the dog (5.24) at low loads and approximately equal at higher loads (1.63 and 1.39, respectively). The computational and experimental analyses showed that the larger shear strain and more nonuniform in-plane extension in the rat may be an indication of significantly different anisotropic material properties in these two species, and implies differences in the collagen ultrastructure.

Stress and strain as regulators of myocardial growth

The response of the heart to altered hemodynamic loading is growth or remodeling of myocytes and the extracellular matrix. In order to describe and mathematically model this dynamic and complex system of growing and resorbing tissue, the stimulating factor for tissue growth must be found, and up to now is not known. Most evidence, both in tissue and at the cellular level, points to a mechanical factor as the stimulus, and most likely a deformation signal is transduced to initiate protein synthesis. At the cellular level mechanotransduction likely takes place at the cellular membrane, although multiple biochemical and mechanical pathways have been proposed which induce transcription in the nucleus and eventual protein upregulation. The results of a recent mathematical analysis based on experimental data suggest that end-diastolic fiber strain at the tissue level may be the stimulus to one mode of tissue growth: volume-overload hypertrophy. This is the only mechanical factor that we found to be normalized after volume overload hypertrophy. But other studies do not agree with this result, and other modes of hypertrophy may be regulated by different factors or combinations of factors.