Increased myocyte calcium sensitivity in end-stage pediatric dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure (HF) in children, resulting in high mortality and need for heart transplantation. The pathophysiology underlying pediatric DCM is largely unclear; however, there is emerging evidence that molecular adaptations and response to conventional HF medications differ between children and adults. To gain insight into alterations leading to systolic dysfunction in pediatric DCM, we measured cardiomyocyte contractile properties and sarcomeric protein phosphorylation in explanted pediatric DCM myocardium (N = 8 subjects) compared with nonfailing (NF) pediatric hearts (N = 8 subjects). Force-pCa curves were generated from skinned cardiomyocytes in the presence and absence of protein kinase A. Sarcomeric protein phosphorylation was quantified with Pro-Q Diamond staining after gel electrophoresis. Pediatric DCM cardiomyocytes demonstrate increased calcium sensitivity (pCa50 =5.70 ± 0.0291), with an associated decrease in troponin (Tn)I phosphorylation compared with NF pediatric cardiomyocytes (pCa50 =5.59 ± 0.0271, P = 0.0073). Myosin binding protein C and TnT phosphorylation are also lower in pediatric DCM, whereas desmin phosphorylation is increased. Pediatric DCM cardiomyocytes generate peak tension comparable to that of NF pediatric cardiomyocytes [DCM 29.7 mN/mm2, interquartile range (IQR) 21.5-49.2 vs. NF 32.8 mN/mm2, IQR 21.5-49.2 mN/mm2; P = 0.6125]. In addition, cooperativity is decreased in pediatric DCM compared with pediatric NF (Hill coefficient: DCM 1.56, IQR 1.31-1.94 vs. NF 1.94, IQR 1.36-2.86; P = 0.0425). Alterations in sarcomeric phosphorylation and cardiomyocyte contractile properties may represent an impaired compensatory response, contributing to the detrimental DCM phenotype in children.NEW & NOTEWORTHY Our study is the first to demonstrate that cardiomyocytes from infants and young children with dilated cardiomyopathy (DCM) exhibit increased calcium sensitivity (likely mediated by decreased troponin I phosphorylation) compared with nonfailing pediatric cardiomyocytes. Compared with published values in adult cardiomyocytes, pediatric cardiomyocytes have notably decreased cooperativity, with a further reduction in the setting of DCM. Distinct adaptations in cardiomyocyte contractile properties may contribute to a differential response to pharmacological therapies in the pediatric DCM population.

Sexually dimorphic myofilament function and cardiac troponin I phosphospecies distribution in hypertrophic cardiomyopathy mice.

The pathological progression of hypertrophic cardiomyopathy (HCM) is sexually dimorphic such that male HCM mice develop phenotypic indicators of cardiac disease well before female HCM mice. Here, we hypothesized that alterations in myofilament function underlies, in part, this sex dimorphism in HCM disease development. Firstly, 10-12month female HCM (harboring a mutant [R403Q] myosin heavy chain) mice presented with proportionately larger hearts than male HCM mice. Next, we determined Ca(2+)-sensitive tension development in demembranated cardiac trabeculae excised from 10-12month female and male HCM mice. Whereas HCM did not impact Ca(2+)-sensitive tension development in male trabeculae, female HCM trabeculae were more sensitive to Ca(2+) than wild-type (WT) counterparts and both WT and HCM males. We hypothesized that the underlying cause of this sex difference in Ca(2+)-sensitive tension development was due to changes in Ca(2+) handling and sarcomeric proteins, including expression of SR Ca(2+) ATPase (2a) (SERCA2a), ß-myosin heavy chain (ß-MyHC) and post-translational modifications of myofilament proteins. Female HCM hearts showed an elevation of SERCA2a and ß-MyHC protein whereas male HCM hearts showed a similar elevation of ß-MyHC protein but a reduced level of cardiac troponin T (cTnT) phosphorylation. We also measured the distribution of cardiac troponin I (cTnI) phosphospecies using phosphate-affinity SDS-PAGE. The distribution of cTnI phosphospecies depended on sex and HCM. In conclusion, female and male HCM mice display sex dimorphic myofilament function that is accompanied by a sex- and HCM-dependent distribution of sarcomeric proteins and cTnI phosphospecies.

Alignment of multi-layered muscle cells within three-dimensional hydrogel macrochannels.

This work describes the development and testing of poly(ethylene glycol) (PEG) hydrogels with independently controlled dimensions of wide and deep macrochannels for their ability to promote alignment of skeletal myoblasts and myoblast differentiation. A UV-photopatterned thiol-ene mold was employed to produce long channels, which ranged from ∼40 to 200 µm in width and from ∼100 to 200 µm in depth, within a PEG-RGD hydrogel. Skeletal myoblasts (C2C12) were successfully cultured multiple cell layers deep within the channels. Decreasing channel width, increasing channel depth and, interestingly, increasing cell layer away from the channel base all contributed to a decreased interquartile range of cell angle relative to the long axis of the channel wall, indicating improved cell alignment. Differentiation of skeletal myoblasts into myotubes was confirmed by gene expression for myoD, myogenin and MCH IIb, and myotube formation for all channel geometries, but was not dependent on channel size. Qualitatively, myotubes were characteristically different, as myotubes were larger and had more nuclei in larger channels. Overall, our findings demonstrate that relatively large features, which do not readily facilitate cell alignment in two dimensions, promote cell alignment when presented in three dimensions, suggesting an important role for three-dimensional spatial cues.

Biochemical and myofilament responses of the right ventricle to severe pulmonary hypertension.

Right ventricular (RV) failure is one of the strongest predictors of mortality both in the presence of left ventricular decompensation and in the context of pulmonary vascular disease. Despite this, there is a limited understanding of the biochemical and mechanical characteristics of the pressure-overloaded RV at the level of the cardiac myocyte. To better understand this, we studied ventricular muscle obtained from neonatal calves that were subjected to hypobaric atmospheric conditions, which result in profound pulmonary hypertension. We found that RV pressure overload resulted in significant changes in the phosphorylation of key contractile proteins. Total phosphorylation of troponin I was decreased with pressure overload, predominantly reflecting changes at the putative PKA site at Ser(22/23). Similarly, both troponin T and myosin light chain 2 showed a significant decline in phosphorylation. Desmin was unchanged, and myosin-binding protein C (MyBP-C) phosphorylation was apparently increased. However, the apparent increase in MyBP-C phosphorylation was not due to phosphorylation but rather to an increase in MyBP-C total protein. Importantly, these findings were seen in all regions of the RV and were paralleled by reduced Ca(2+) sensitivity with preserved maximal Ca(2+) saturated developed force normalized to cross-sectional area in isolated skinned right ventricular myocyte fragments. No changes in total force or cooperativity were seen. Taken together, these results suggest that RV failure is mechanistically unique from left ventricular failure.

Incomplete recovery of myocyte contractile function despite improvement of myocardial architecture with left ventricular assist device support.

BACKGROUND: Unloading a failing heart with a left ventricular assist device (LVAD) can improve ejection fraction (EF) and LV size; however, recovery with LVAD explantation is rare. We hypothesized that evaluation of myocyte contractility and biochemistry at the sarcomere level before and after LVAD may explain organ-level changes. METHODS AND RESULTS: Paired LV tissue samples were frozen from 8 patients with nonischemic cardiomyopathy at LVAD implantation (before LVAD) and before cardiac transplantation (after LVAD). These were compared with 8 nonfailing hearts. Isolated skinned myocytes were purified and attached to a force transducer, and dimensions, maximum calcium-saturated force, calcium sensitivity, and myofilament cooperativity were assessed. Relative isoform abundance and phosphorylation levels of sarcomeric contractile proteins were measured. With LVAD support, the unloaded EF improved (10.0±1.0% to 25.6±11.0%, P=0.007), LV size decreased (LV internal dimension at end diastole, 7.6±1.2 to 4.9±1.4 cm; P<0.001), and myocyte dimensions decreased (cross-sectional area, 1247±346 to 638±254 µm(2); P=0.001). Maximum calcium-saturated force improved after LVAD (3.6±0.9 to 7.3±1.8 mN/mm(2), P<0.001) implantation but was still lower than in nonfailing hearts (7.3±1.8 versus 17.6±1.8 mN/mm(2), P<0.001). An increase in troponin I (TnI) phosphorylation after LVAD implantation was noted, but protein kinase C phosphorylation of TnI decreased. Biochemical changes of other sarcomeric proteins were not observed after LVAD. CONCLUSIONS: There is significant improvement in LV and myocyte size with LVAD, but there is only partial recovery of EF and myocyte contractility. LVAD support was associated only with biochemical changes in TnI, suggesting that alternate mechanisms might contribute to contractile changes after LVAD and that additional interventions may be needed to alter biochemical remodeling of the sarcomere to further enhance myofilament and organ-level recovery.

Protein kinase A changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils.

We investigated the effect of PKA treatment (1 U/ml) on the mechanical properties of isolated human cardiac myofibrils. PKA treatment was associated with significant incorporation of radiolabeled phosphate into several sarcomeric proteins including troponin I and myosin binding protein C and was also associated with a right shift in the tension-pCa relation (ΔpCa(50) = 0.2 ± 0.1). PKA treatment also caused right shifts in the pCa dependence of the rate of tension development, tension redevelopment, and the linear and exponential phases of myofibril relaxation. However, there was no change in the same measures of crossbridge turnover when expressed as a function of tension. We conclude that the changes in crossbridge kinetics as a function of calcium concentration reflect a reduced tension due to a lower calcium sensitivity and that the relationship between crossbridge kinetics and tension was unchanged, indicating no direct effect of PKA treatment on crossbridge cycling.

Tissue procurement strategies affect the protein biochemistry of human heart samples.

The ability to analyze the biochemical properties of human cardiac tissue is critical both to an understanding of cardiac pathology and also to the development of novel pharmacotherapies. However current strategies for tissue procurement are not uniform and are potentially biased. In this study we contrasted several commonly used approaches for tissue sampling in order to determine their impact on contractile protein biochemistry. Not surprisingly our results show that different tissue handling strategies have the potential to produce a wide variation in the phosphorylation and proteolysis of selected contractile proteins. However this was not uniform: phosphorylation of troponin I (TnI) and myosin light chain 2 (MLC2) varied significantly depending on approach whereas changes in desmin and myosin binding protein C (MyBP-C) were relatively unaffected. Moreover, some strategies increased whereas others reduced TnI phosphorylation, suggesting a dynamic balance between kinase and phosphatase activities. Overall, procurement strategies that involved maintenance of tissue in cardioplegia solution deviated most dramatically from prompt and rapid tissue immersion in liquid nitrogen.

Analysing force-pCa curves.

We investigated three forms of the Hill equation used to fit force-calcium data from skinned muscle experiments; Two hyperbolic forms that relate force to calcium concentration directly, and a sigmoid form that relates force to the -log(10) of the calcium concentration (pCa). The equations were fit to force-calcium data from 39 cardiac myocytes (up to five myocytes from each of nine mice) and the Hill coefficient and the calcium required for half maximal activation, expressed as a concentration (EC(50)) and as a pCa value (pCa(50)) were obtained. The pCa(50) values were normally distributed and the EC(50) values were found to approximate a log-normal distribution. Monte Carlo simulations confirmed that these distributions were intrinsic to the Hill equation. Statistical tests such as the t-test are robust to moderate levels of departure from normality as seen here, and either EC(50) or pCa(50) may be used to test for significant differences so long as it is kept in mind that ΔEC50 is an additive measure of change and that ΔpCa50 is a ratiometric measure of change. The Hill coefficient was found to be sufficiently log-normally distributed that log-transformed values should be used to test for statistically significant differences.

AFos inhibits phenylephrine-mediated contractile dysfunction by altering phospholamban phosphorylation.

Using neonatal rat ventricular myocytes, we previously reported that the expression of a dominant negative form of the c-Fos proto-oncogene (AFos) inhibited activator protein 1 activity and blocked the induction of the pathological gene profile stimulated by phenylephrine (PE) while leaving growth unaffected. We now extend these observations to the adult rat ventricular myocyte (ARVM) to understand the relationship between gene expression, growth, and function. Ventricular myocytes were isolated from adult rats and infected with adenovirus expressing beta-galactosidase (control) or AFos. The cells were subsequently treated with PE, and protein synthesis, gene program, calcium transients, and contractility were evaluated. As seen with the neonatal rat ventricular myocytes, in control cells PE stimulated an increase in protein synthesis, induced the pathological gene profile, and exhibited both depressed contractility and calcium transients. Although ARVMs expressing AFos still had PE-induced growth, pathological gene expression as well as contractility and calcium handling abnormalities were inhibited. To determine a possible mechanism of the preserved myocyte function in AFos-expressing cells, we examined phospholamban (PLB) and sarco(endo)plasmic reticulum calcium-ATPase proteins. Although there was no change in total PLB or sarco(endo)plasmic reticulum calcium-ATPase expression in response to PE treatment, PE decreased the phosphorylation of PLB at serine-16, an observation that was prevented in AFos-expressing cells. In conclusion, although PE-induced growth was unaffected in AFos-expressing ARVMs, the expression of the pathological gene profile was inhibited and both contractile function and calcium cycling were preserved. The inhibition of functional deterioration was, in part, due to the preservation of PLB phosphorylation.

Stage-specific changes in myofilament protein phosphorylation following myocardial infarction in mice.

The response of cardiac muscle to an insult such as myocardial infarction includes changes in the expression of numerous signaling proteins and modulation of gene expression, as well as post-translational modifications of existing proteins. Most studies to date have defined these in end-stage cardiac muscle thus obviating consideration of the temporal progression that causes the heart to transition from a compensated to a decompensated phenotype. To explore these transitions, we examined contractile protein biochemistry in a mouse MI model at two early time points: 2 days and 2 weeks post-infarct and at two later time points: 2 and 4 months post-infarct. Phosphorylation of myofilament proteins was analyzed using phosphospecific staining of polyacrylamide gels, and whenever possible, phosphospecific antibodies. Phosphorylation of myosin binding protein c, the myosin regulatory light chain and troponin I were all decreased relative to sham operated animals at both early time points. However, by 2 months, total phosphorylation of all the major myofilament proteins normalized and at both 2 and 4 months, there was a significant increase in troponin I phosphorylation. One-dimensional IEF of troponin I coupled with phospho-specific antibody analysis demonstrated a redistribution of phosphorylation sites with a significant initial decline at the putative PKA sites, Serine 22,23, and a subsequent increase at the putative PKC site, serine 43,45. These data suggest that temporal changes in myofilament protein phosphorylation contribute both to the initial compensatory hyperdynamic response to myocardial infarction and subsequently to the gradual progression to myocardial failure.

Glass microneedles for force measurements: a finite-element analysis model.

Changes in developed force (0.1-3.0 microN) observed during contraction of single myofibrils in response to rapidly changing calcium concentrations can be measured using glass microneedles. These microneedles are calibrated for stiffness and deflect on response to developed myofibril force. The precision and accuracy of kinetic measurements are highly dependent on the structural and mechanical characteristics of the microneedles, which are generally assumed to have a linear force-deflection relationship. We present a finite-element analysis (FEA) model used to simulate the effects of measurable geometry on stiffness as a function of applied force and validate our model with actual measured needle properties. In addition, we developed a simple heuristic constitutive equation that best describes the stiffness of our range of microneedles used and define limits of geometry parameters within which our predictions hold true. Our model also maps a relation between the geometry parameters and natural frequencies in air, enabling optimum parametric combinations for microneedle fabrication that would reflect more reliable force measurement in fluids and physiological environments. We propose a use for this model to aid in the design of microneedles to improve calibration time, reproducibility, and precision for measuring myofibrillar, cellular, and supramolecular kinetic forces.

The troponin C G159D mutation blunts myofilament desensitization induced by troponin I Ser23/24 phosphorylation.

Striated muscle contraction is regulated by the binding of Ca(2+) to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, beta-adrenergic stimulation induces protein kinase A phosphorylation of cardiac troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the troponin complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca(2+)-dependent tension and ATPase rate in whole troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca(2+) in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca(2+) sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca(2+)-sensitive tension development without altering cross-bridge cycling. Measurements in purified troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca(2+)-binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on troponin Ca(2+)-binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through beta-adrenergic signaling as well as the development of cardiomyopathy.

Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics.

We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC(50)) and whether modulation of EC(50) affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20 degrees C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC(50); ssTnI-chimera Tn exchange induced a further decrease in EC(50) (in microM: endogenous Tn, 1.35 +/- 0.08; cTnI, 1.04 +/- 0.13; ssTnI-chimera Tn, 0.47 +/- 0.03). EC(50) was also decreased by application of 100 microM bepridil (control: 2.04 +/- 0.03 microM; bepridil 1.35 +/- 0.03 microM). Maximum tension was not different between any groups. Despite marked alterations in EC(50), none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca(2+) sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC(50). This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.

The effect of myosin regulatory light chain phosphorylation on the frequency-dependent regulation of cardiac function.

Although it has been suggested that in cardiac muscle the phosphorylation level of myosin regulatory light chain (RLC) correlates with frequency of stimulation, its significance in the modulation of the force-frequency and pressure-frequency relationships remains unclear. We examined the role of RLC phosphorylation on the force-frequency relation (papillary muscles), the pressure-frequency relation (Langendorff perfused hearts) and shortening-frequency relation (isolated cardiac myocytes) in nontransgenic (NTG) and transgenic mouse hearts expressing a nonphosphorylatable RLC protein (RLC(P-)). At 22 degrees C, NTG and RLC(P-) muscles showed a negative force-frequency relation. At 32 degrees C, at frequencies above 1 Hz, both groups showed a flat force-frequency relation. There was a small increase in RLC phosphorylation in NTG muscles when the frequency of stimulation was increased from 0.2 Hz to 4.0 Hz. However, the level of RLC phosphorylation in these isolated muscles was significantly lower compared to samples taken from NTG intact hearts. In perfused hearts, there was no difference in the slope of pressure-frequency relationship between groups, but the RLC(P-) group consistently developed a reduced systolic pressure and demonstrated a decreased contractility. There was no difference in the level of RLC phosphorylation in hearts paced at 300 and 600 bpm. In RLC(P-) hearts, the level of TnI phosphorylation was reduced compared to NTG. There was no change in the expression of PLB between groups, but expression of SERCA2 was increased in hearts from RLC(P-) compared to NTG. In isolated cardiac myocytes, there was no change in shortening-frequency relationship between groups. Moreover, there was no change in Ca(2+) transient parameters in cells from NTG and RLC(P-) hearts. Our data demonstrate that in cardiac muscle RLC phosphorylation is not an essential determinant of force- and pressure-frequency relations but the absence of RLC phosphorylation decreases contractility in force/pressure developing preparations.

Impact of osmotic compression on sarcomere structure and myofilament calcium sensitivity of isolated rat myocardium.

Changes in interfilament lattice spacing have been proposed as the mechanism underlying myofilament length-dependent activation. Much of the evidence to support this theory has come from experiments in which high-molecular-weight compounds, such as dextran, were used to osmotically shrink the myofilament lattice. However, whether interfilament spacing directly affects myofilament calcium sensitivity (EC(50)) has not been established. In this study, skinned isolated rat myocardium was osmotically compressed over a wide range (Dextran T500; 0-6%), and EC(50) was correlated to both interfilament spacing and I(1,1)/I(1,0) intensity ratio. The latter two parameters were determined by X-ray diffraction in a separate group of skinned muscles. Osmotic compression induced a marked reduction in myofilament lattice spacing, concomitant with increases in both EC(50) and I(1,1)/I(1,0) intensity ratio. However, interfilament spacing was not well correlated with EC(50) (r(2) = 0.78). A much better and deterministic relationship was observed between EC(50) and the I(1,1)/I(1,0) intensity ratio (r(2) = 0.99), albeit with a marked discontinuity at low levels of dextran compression; that is, a small amount of external osmotic compression (0.38 kPa, corresponding to 1% Dextran T500) produced a stepwise increase in the I(1,1)/I(1,0) ratio concomitant with a stepwise decrease in EC(50). These parameters then remained stable over a wide range of further applied osmotic compression (up to 6% dextran). These findings provide support for a "switch-like" activation mechanism within the cardiac sarcomere that is highly sensitive to changes in external osmotic pressure.

Absence of force suppression in rabbit bladder correlates with low expression of heat shock protein 20.

BACKGROUND: Nitroglycerin can induce relaxation of swine carotid artery without sustained reductions in [Ca2+]i or myosin regulatory light chain (MRLC) phosphorylation. This has been termed force suppression and been found to correlate with ser16-phosphorylation of heat shock protein 20 (HSP20). We tested for the existence of this mechanism in a smooth muscle that is not responsive to nitric oxide. METHODS: Isometrically mounted mucosa free rabbit bladder strips were contracted with carbachol and relaxed with 8-Br-cGMP, forskolin, or isoprenaline. RESULTS: Contraction was associated with a highly cooperative relation between MRLC phosphorylation and force such that very small increases in MRLC phosphorylation induced large increases in force. Relaxation induced by 8-Br-cGMP, forskolin, or isoprenaline did not shift the MRLC phosphorylation-force relation from that observed with carbachol alone, i.e. there was no force suppression. HSP20 content was negligible (approximately two hundred-fold less than swine carotid). CONCLUSION: The lack of force suppression in the absence of HSP20 is consistent with the hypothesized role for HSP20 in the force suppression observed in tonic smooth muscles.

Kymograph: a muscle physiology toolkit.

Kymograph is a freely available, Linux-based data acquisition and control system that is designed as a device driver and a series of scriptable tools. It allows the creation of scripts for performing a variety of measurements related to muscle physiology. It can control hardware (2 analog channels) and capture data (up to 16 channels) at rates of up to 1 kHz. It also has the ability to generate stimulus control pulses with a resolution of 10 micros. The tools can be used with any of the common scripting languages to generate complete control and capture routines for performing measurements such as length-tension curves or force-velocity relationships. Source code is provided to allow easy extension of the software.