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 ( = 8 subjects) compared with nonfailing (NF) pediatric hearts ( = 8 subjects).

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.