The embryonic myosin R672C mutation that underlies Freeman-Sheldon syndrome impairs cross-bridge detachment and cycling in adult skeletal muscle.

Distal arthrogryposis is the most common known heritable cause of congenital contractures (e.g. clubfoot) and results from mutations in genes that encode proteins of the contractile complex of skeletal muscle cells. Mutations are most frequently found in MYH3 and are predicted to impair the function of embryonic myosin. We measured the contractile properties of individual skeletal muscle cells and the activation and relaxation kinetics of isolated myofibrils from two adult individuals with an R672C substitution in embryonic myosin and distal arthrogryposis syndrome 2A (DA2A) or Freeman-Sheldon syndrome. In R672C-containing muscle cells, we observed reduced specific force, a prolonged time to relaxation and incomplete relaxation (elevated residual force). In R672C-containing muscle myofibrils, the initial, slower phase of relaxation had a longer duration and slower rate, and time to complete relaxation was greatly prolonged. These observations can be collectively explained by a small subpopulation of myosin cross-bridges with greatly reduced detachment kinetics, resulting in a slower and less complete deactivation of thin filaments at the end of contractions. These findings have important implications for selecting and testing directed therapeutic options for persons with DA2A and perhaps congenital contractures in general.

Prevascularized microtemplated fibrin scaffolds for cardiac tissue engineering applications.

Myocardial infarction (MI) causes significant cell loss and damage to myocardium. Cell-based therapies for treatment of MI aim to remuscularize the resultant scar tissue, but the majority of transplanted cells do not survive or integrate with the host tissue. Scaffolds can improve cell retention following construct implantation, but often do little to enhance host-graft integration and/or show limited biodegradation. Fibrin is an ideal biomaterial for cardiac tissue engineering as it is a natural, biodegradable polymer that can induce neovascularization, promote cell attachment, and has tunable mechanical properties. Here we describe a novel, high-density microtemplated fibrin scaffold seeded with a tri-cell mixture of cardiomyocytes, endothelial cells (ECs), and fibroblasts to mimic native cardiac tissue in structure and cellular composition to improve cell retention and promote integration with the host tissue. Scaffolds were designed with uniform architecture of parallel 60 µm microchannels surrounded by an interconnected microporous network of 27-µm-diameter pores and mechanical stiffness comparable to native cardiac tissues (70-90kPa). Scaffold degradation was controlled with the addition of Factor XIII (FXIII) and/or protease inhibitor (aprotinin). Unmodified scaffolds had a fast degradation profile both in vitro (19.9%±3.9% stiffness retention after 10 days) and in vivo. Scaffolds treated with FXIII showed an intermediate degradation profile in vitro (45.8%±5.9%), while scaffolds treated with aprotinin or both FXIII and aprotinin showed significantly slowed degradation in vitro (60.9%±5.2% and 76.4%±7.6%, respectively, p<0.05). Acellular aprotinin scaffold myocardial implants showed decreased collagen deposition after 7 days. Unmodified and aprotinin implants could not be located by 14 days, while 2 of 8 FXIII implants were found, but were significantly degraded. Constructs supported seeded cell survival and organization in vitro, promoting EC-lined lumen structure formation in construct channels and colocalization of viable ECs and cardiomyocytes. In addition, constructs promoted extracellular matrix deposition by seeded cells, as shown by collagen staining within construct channels and by significant increases in construct stiffness over 10 days in vitro (209%±32%, p<0.05). The data suggest our fibrin scaffolds are ideally designed to promote graft cell survival and organization, thus improving chances of promoting construct integration with the host tissue upon implantation.

N-terminal phosphorylation of cardiac troponin-I reduces length-dependent calcium sensitivity of contraction in cardiac muscle.

Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for ß-adrenergic modulation of cardiac contractility. In myofilaments PKA targets troponin I (cTnI), myosin binding protein-C (cMyBP-C) and titin. We studied how this affects the sarcomere length (SL) dependence of force-pCa relations in demembranated cardiac muscle. To distinguish cTnI from cMyBP-C/titin phosphorylation effects on the force-pCa relationship, endogenous troponin (Tn) was exchanged in rat ventricular trabeculae with either wild-type (WT) Tn, non-phosphorylatable cTnI (S23/24A) Tn or phosphomimetic cTnI (S23/24D) Tn. PKA cannot phosphorylate either cTnI S23/24 variant, leaving cMyBP-C/titin as PKA targets. Force was measured at 2.3 and 2.0 µm SL. Decreasing SL reduced maximal force (F(max)) and Ca(2+) sensitivity of force (pCa(50)) similarly with WT and S23/24A trabeculae. PKA treatment of WT and S23/24A trabeculae reduced pCa(50) at 2.3 but not at 2.0 µm SL, thus eliminating the SL dependence of pCa(50). In contrast, S23/24D trabeculae reduced pCa(50) at both SL values, primarily at 2.3 µm, also eliminating SL dependence of pCa(50). Subsequent PKA treatment moderately reduced pCa(50) at both SLs. At each SL, F(max) was unaffected by either Tn exchange and/or PKA treatment. Low-angle X-ray diffraction was performed to determine whether pCa(50) shifts were associated with changes in myofilament spacing (d(1,0)) or thick-thin filament interaction. PKA increased d(1,0) slightly under all conditions. The ratios of the integrated intensities of the equatorial X-ray reflections (I(1,1)/I(1,0)) indicate that PKA treatment increased crossbridge proximity to thin filaments under all conditions. The results suggest that phosphorylation by PKA of either cTnI or cMyBP-C/titin independently reduces the pCa(50) preferentially at long SL, possibly through reduced availability of thin filament binding sites (cTnI) or altered crossbridge recruitment (cMyBP-C/titin). Preferential reduction of pCa(50) at long SL may not reduce cardiac output during periods of high metabolic demand because of increased intracellular Ca(2+) during ß-adrenergic stimulation.

Enhanced Ca2+ binding of cardiac troponin reduces sarcomere length dependence of contractile activation independently of strong crossbridges.

Calcium sensitivity of the force-pCa relationship depends strongly on sarcomere length (SL) in cardiac muscle and is considered to be the cellular basis of the Frank-Starling law of the heart. SL dependence may involve changes in myofilament lattice spacing and/or myosin crossbridge orientation to increase probability of binding to actin at longer SLs. We used the L48Q cardiac troponin C (cTnC) variant, which has enhanced Ca(2+) binding affinity, to test the hypotheses that the intrinsic properties of cTnC are important in determining 1) thin filament binding site availability and responsiveness to crossbridge activation and 2) SL dependence of force in cardiac muscle. Trabeculae containing L48Q cTnC-cTn lost SL dependence of the Ca(2+) sensitivity of force. This occurred despite maintaining the typical SL-dependent changes in maximal force (F(max)). Osmotic compression of preparations at SL 2.0 µm with 3% dextran increased F(max) but not pCa(50) in L48Q cTnC-cTn exchanged trabeculae, whereas wild-type (WT)-cTnC-cTn exchanged trabeculae exhibited increases in both F(max) and pCa(50). Furthermore, crossbridge inhibition with 2,3-butanedione monoxime at SL 2.3 µm decreased F(max) and pCa(50) in WT cTnC-cTn trabeculae to levels measured at SL 2.0 µm, whereas only F(max) was decreased with L48Q cTnC-cTn. Overall, these results suggest that L48Q cTnC confers reduced crossbridge dependence of thin filament activation in cardiac muscle and that changes in the Ca(2+) sensitivity of force in response to changes in SL are at least partially dependent on properties of thin filament troponin.

Transcription factor CHF1/Hey2 regulates EC coupling and heart failure in mice through regulation of FKBP12.6.

Heart failure is a leading cause of morbidity and mortality in Western society. The cardiovascular transcription factor CHF1/Hey2 has been linked to experimental heart failure in mice, but the mechanisms by which it regulates myocardial function remain incompletely understood. The objective of this study was to determine how CHF1/Hey2 affects development of heart failure through examination of contractility in a myocardial knockout mouse model. We generated myocardial-specific knockout mice. At baseline, cardiac function was normal, but, after aortic banding, the conditional knockout mice demonstrated a greater increase in ventricular weight-to-body weight ratio compared with control mice (5.526 vs. 4.664 mg/g) and a significantly decreased ejection fraction (47.8 vs. 72.0% control). Isolated cardiac myocytes from these mice showed decreased calcium transients and fractional shortening after electrical stimulation. To determine the molecular basis for these alterations in excitation-contraction coupling, we first measured total sarcoplasmic reticulum calcium stores and calcium-dependent force generation in isolated muscle fibers, which were normal, suggesting a defect in calcium cycling. Analysis of gene expression demonstrated normal expression of most genes known to be involved in myocardial calcium cycling, with the exception of the ryanodine receptor binding protein FKBP12.6, which was expressed at increased levels in the conditional knockout hearts. Treatment of the isolated knockout myocytes with FK506, which inhibits the association of FKBP12.6 with the ryanodine receptor, restored contractile function. These findings demonstrate that conditional deletion of CHF1/Hey2 in the myocardium leads to abnormalities in calcium handling mediated by FKBP12.6 that predispose to pressure overload-induced heart failure.

Growth of engineered human myocardium with mechanical loading and vascular coculture.

RATIONALE: The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood. OBJECTIVE: We seek to elucidate the responses of immature human myocardium to mechanical load and vascularization using tissue engineering approaches. METHODS AND RESULTS: Using human embryonic stem cell and human induced pluripotent stem cell-derived cardiomyocytes in a 3-dimensional collagen matrix, we show that uniaxial mechanical stress conditioning promotes 2-fold increases in cardiomyocyte and matrix fiber alignment and enhances myofibrillogenesis and sarcomeric banding. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) versus unconditioned constructs. Addition of endothelial cells enhances cardiomyocyte proliferation under all stress conditions (14% to 19%), and addition of stromal supporting cells enhances formation of vessel-like structures by ≈10-fold. Furthermore, these optimized human cardiac tissue constructs generate Starling curves, increasing their active force in response to increased resting length. When transplanted onto hearts of athymic rats, the human myocardium survives and forms grafts closely apposed to host myocardium. The grafts contain human microvessels that are perfused by the host coronary circulation. CONCLUSIONS: Our results indicate that both mechanical load and vascular cell coculture control cardiomyocyte proliferation, and that mechanical load further controls the hypertrophy and architecture of engineered human myocardium. Such constructs may be useful for studying human cardiac development as well as for regenerative therapy.

Cell therapy enhances function of remote non-infarcted myocardium.

Cell transplantation improves cardiac function after myocardial infarction; however, the underlying mechanisms are not well-understood. Therefore, the goals of this study were to determine if neonatal rat cardiomyocytes transplanted into adult rat hearts 1 week after infarction would, after 8-10 weeks: 1) improve global myocardial function, 2) contract in a Ca2+ dependent manner, 3) influence mechanical properties of remote uninjured myocardium and 4) alter passive mechanical properties of infarct regions. The cardiomyocytes formed small grafts of ultrastructurally maturing myocardium that enhanced fractional shortening compared to non-treated infarcted hearts. Chemically demembranated tissue strips of cardiomyocyte grafts produced force when activated by Ca2+, whereas scar tissue did not. Furthermore, the Ca2+ sensitivity of force was greater in cardiomyocyte grafts compared to control myocardium. Surprisingly, cardiomyocytes grafts isolated in the infarct zone increased Ca2+ sensitivity of remote uninjured myocardium to levels greater than either remote myocardium from non-treated infarcted hearts or sham-operated controls. Enhanced calcium sensitivity was associated with decreased phosphorylation of cTnT, tropomyosin and MLC2, but not changes in myosin or troponin isoforms. Passive compliance of grafts resembled normal myocardium, while infarct tissue distant from grafts had compliance typical of scar. Thus, cardiomyocyte grafts are contractile, improve local tissue compliance and enhance calcium sensitivity of remote myocardium. Because the volume of remote myocardium greatly exceeds that of the grafts, this enhanced calcium sensitivity may be a major contributor to global improvements in ventricular function after cell transplantation.

Sarcomere length dependence of rat skinned cardiac myocyte mechanical properties: dependence on myosin heavy chain.

The effects of sarcomere length (SL) on sarcomeric loaded shortening velocity, power output and rates of force development were examined in rat skinned cardiac myocytes that contained either alpha-myosin heavy chain (alpha-MyHC) or beta-MyHC at 12 +/- 1 degrees C. When SL was decreased from 2.3 microm to 2.0 microm submaximal isometric force decreased approximately 40% in both alpha-MyHC and beta-MyHC myocytes while peak absolute power output decreased 55% in alpha-MyHC myocytes and 70% in beta-MyHC myocytes. After normalization for the fall in force, peak power output decreased about twice as much in beta-MyHC as in alpha-MyHC myocytes (41% versus 20%). To determine whether the fall in normalized power was due to the lower force levels, [Ca(2+)] was increased at short SL to match force at long SL. Surprisingly, this led to a 32% greater peak normalized power output at short SL compared to long SL in alpha-MyHC myocytes, whereas in beta-MyHC myocytes peak normalized power output remained depressed at short SL. The role that interfilament spacing plays in determining SL dependence of power was tested by myocyte compression at short SL. Addition of 2% dextran at short SL decreased myocyte width and increased force to levels obtained at long SL, and increased peak normalized power output to values greater than at long SL in both alpha-MyHC and beta-MyHC myocytes. The rate constant of force development (k(tr)) was also measured and was not different between long and short SL at the same [Ca(2+)] in alpha-MyHC myocytes but was greater at short SL in beta-MyHC myocytes. At short SL with matched force by either dextran or [Ca(2+)], k(tr) was greater than at long SL in both alpha-MyHC and beta-MyHC myocytes. Overall, these results are consistent with the idea that an intrinsic length component increases loaded crossbridge cycling rates at short SL and beta-MyHC myocytes exhibit a greater sarcomere length dependence of power output.

Porcine cardiac myocyte power output is increased after chronic exercise training.

Chronic exercise training increases the functional capacity of the heart, perhaps by increased myocyte contractile function, as has been observed in rodent exercise models. We examined whether cardiac myocyte function is enhanced after chronic exercise training in Yucatan miniature swine, whose heart characteristics are similar to humans. Animals were designated as either sedentary (Sed), i.e., cage confined, or exercise trained (Ex), i.e., underwent 16-20 wk of progressive treadmill training. Exercise training efficacy was shown with significantly increased heart weight-to-body weight ratios, skeletal muscle citrate synthase activity, and exercise tolerance. Force-velocity properties were measured by attaching skinned cardiac myocytes between a force transducer and position motor, and shortening velocities were measured over a range of loads during maximal Ca2+ activation. Myocytes (n = 9) from nine Ex pigs had comparable force production but a approximately 30% increase in peak power output compared with myocytes (n = 8) from eight Sed. Interestingly, Ex myofibrillar samples also had higher baseline PKA-induced phosphorylation levels of cardiac troponin I, which may contribute to the increase in power. Overall, these results suggest that enhanced power-generating capacity of porcine cardiac myofibrils contributes to improved cardiac function after chronic exercise training.

Power output is linearly related to MyHC content in rat skinned myocytes and isolated working hearts.

The amount of work the heart can perform during ejection is governed by the inherent contractile properties of individual myocytes. One way to alter contractile properties is to alter contractile proteins such as myosin heavy chain (MyHC), which is known to demonstrate isoform plasticity in response to disease states. The purpose of this study was to examine myocyte functionality over the complete range of MyHC expression in heart, from 100% alpha-MyHC to 100% beta-MyHC, using euthyroid and hypothyroid rats. Peak power output in skinned cardiac myocytes decreased as a nearly linear function of beta-MyHC expression during maximal (r2 = 0.85, n = 44 myocyte preparations) and submaximal (r2 = 0.82, n = 31 myocyte preparations) Ca2+ activation. To determine whether single myocyte function translated to the level of the whole heart, power output was measured in working heart preparations expressing varied ratios of MyHC. Left ventricular power output of isolated working heart preparations also decreased as a linear function of increasing beta-MyHC expression (r2 = 0.82, n = 34 myocyte preparations). These results demonstrate that power output is highly dependent on MyHC expression in single myocytes, and this translates to the performance of working left ventricles.

Exercise improves impaired ventricular function and alterations of cardiac myofibrillar proteins in diabetic dyslipidemic pigs.

Chronic diabetes is often associated with cardiomyopathy, which may result, in part, from defects in cardiac muscle proteins. We investigated whether a 20-wk porcine model of diabetic dyslipidemia (DD) would impair in vivo myocardial function and yield alterations in cardiac myofibrillar proteins and whether endurance exercise training would improve these changes. Myocardial function was depressed in anesthetized DD pigs (n = 12) compared with sedentary controls (C; n = 13) as evidenced by an approximately 30% decrease in left ventricular fractional shortening and an approximately 35% decrease in +dP/dt measured by noninvasive echocardiography and direct cardiac catheterization, respectively. This depression in myocardial function was improved with chronic exercise as treadmill-trained DD pigs (DDX) (n = 13) had significantly greater fractional shortening and +dP/dt than DD animals. Interestingly, the isoform expression pattern of the myofibrillar regulatory protein, cardiac troponin T (cTnT), was significantly shifted from cTnT1 toward cTnT2 and cTnT3 in DD pigs. Furthermore, this change in cTnT isoform expression pattern was prevented in DDX pigs. Finally, there was a decrease in baseline levels of cAMP-dependent protein kinase-induced phosphorylation of the myofibrillar proteins troponin I and myosin-binding protein-C in DD animals. Overall, these results indicate that 20 wk of DD lead to myocardial dysfunction coincident with significant alterations in myofibrillar proteins, both of which are prevented with endurance exercise training, implying that changes in myofibrillar proteins may contribute, at least in part, to cardiac dysfunction associated with diabetic cardiomyopathy.

Loaded shortening, power output, and rate of force redevelopment are increased with knockout of cardiac myosin binding protein-C.

Myosin binding protein-C (MyBP-C) is localized to the thick filaments of striated muscle where it appears to have both structural and regulatory functions. Importantly, mutations in the cardiac MyBP-C gene are associated with familial hypertrophic cardiomyopathy. The purpose of this study was to examine the role that MyBP-C plays in regulating force, power output, and force development rates in cardiac myocytes. Skinned cardiac myocytes from wild-type (WT) and MyBP-C knockout (MyBP-C-/-) mice were attached between a force transducer and position motor. Force, loaded shortening velocities, and rates of force redevelopment were measured during both maximal and half-maximal Ca2+ activations. Isometric force was not different between the two groups with force being 17.0+/-7.2 and 20.5+/-3.1 kN/m2 in wild-type and MyBP-C-/- myocytes, respectively. Peak normalized power output was significantly increased by 26% in MyBP-C-/- myocytes (0.15+/-0.01 versus 0.19+/-0.03 P/Po x ML/sec) during maximal Ca2+ activations. Interestingly, peak power output in MyBP-C-/- myocytes was increased to an even greater extent (46%, 0.09+/-0.03 versus 0.14+/-0.02 P/Po x ML/sec) during half-maximal Ca2+ activations. There was also an effect on the rate constant of force redevelopment (ktr) during half-maximal Ca2+ activations, with ktr being significantly greater in MyBP-C-/- myocytes (WT=5.8+/-0.9 s(-1) versus MyBP-C-/-=7.7+/-1.7 s(-1)). These results suggest that cMyBP-C is an important regulator of myocardial work capacity whereby MyBP-C acts to limit power output.