Dissociation of structural and functional phenotypes in cardiac myosin-binding protein C conditional knockout mice.

BACKGROUND: Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric protein that dynamically regulates thick-filament structure and function. In constitutive cMyBP-C knockout (cMyBP-C(-/-)) mice, loss of cMyBP-C has been linked to left ventricular dilation, cardiac hypertrophy, and systolic and diastolic dysfunction, although the pathogenesis of these phenotypes remains unclear. METHODS AND RESULTS: We generated cMyBP-C conditional knockout (cMyBP-C-cKO) mice expressing floxed cMyBP-C alleles and a tamoxifen-inducible Cre-recombinase fused to 2 mutated estrogen receptors to study the onset and progression of structural and functional phenotypes caused by the loss of cMyBP-C. In adult cMyBP-C-cKO mice, knockdown of cMyBP-C over a 2-month period resulted in a corresponding impairment of diastolic function and a concomitant abbreviation of systolic ejection, although contractile function was largely preserved. No significant changes in cardiac structure or morphology were immediately evident; however, mild hypertrophy developed after near-complete knockdown of cMyBP-C. In response to pressure overload induced by transaortic constriction, cMyBP-C-cKO mice treated with tamoxifen also developed greater cardiac hypertrophy, left ventricular dilation, and reduced contractile function. CONCLUSIONS: These results indicate that myocardial dysfunction is largely caused by the removal of cMyBP-C and occurs before the onset of cytoarchitectural remodeling in tamoxifen-treated cMyBP-C-cKO myocardium. Moreover, near ablation of cMyBP-C in adult myocardium primarily leads to the development of hypertrophic cardiomyopathy in contrast to the dilated phenotype evident in cMyBP-C(-/-) mice, which highlights the importance of additional factors such as loading stress in determining the expression and progression of cMyBP-C-associated cardiomyopathy.

Cooperative mechanisms underlie differences in myocardial contractile dynamics between large and small mammals.

Ca2+ binding to troponin C (TnC) and myosin cross-bridge binding to actin act in a synergistic cooperative manner to modulate myocardial contraction and relaxation. The responsiveness of the myocardial thin filament to the activating effects of Ca2+ and myosin cross-bridge binding has been well-characterized in small mammals (e.g., mice). Given the nearly 10-fold difference in resting heart rates and twitch kinetics between small and large mammals, it is unlikely that the cooperative mechanisms underlying thin filament activation are identical in these two species. To test this idea, we measured the Ca2+ dependencies of steady-state force and the rate constant of force redevelopment (ktr) in murine and porcine permeabilized ventricular myocardium. While murine myocardium exhibited a steep activation-dependence of ktr, the activation-dependent profile of ktr was significantly reduced in porcine ventricular myocardium. Further insight was attained by examining force-pCa and ktr-pCa relationships. In the murine myocardium, the pCa50 for ktr was right-shifted compared with the pCa50 for force, meaning that increases in steady-state force occurred well before increases in the rate of force redevelopment were observed. In the porcine myocardium, we observed a tighter coupling of the force-pCa and ktr-pCa relationships, as evidenced by near-maximal rates of force redevelopment at low levels of Ca2+ activation. These results demonstrate that the molecular mechanisms underlying the cooperative activation of force are a dynamic property of the mammalian heart, involving, at least in part, the species- and tissue-specific expression of cardiac myosin heavy chain isoforms.

Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the thick filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca(2+)-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.

Magnitude of length-dependent changes in contractile properties varies with titin isoform in rat ventricles.

The effects of differential expression of titin isoforms on sarcomere length (SL)-dependent changes in passive force, maximum Ca(2+)-activated force, apparent cooperativity in activation of force (n(H)), Ca(2+) sensitivity of force (pCa(50)), and rate of force redevelopment (k(tr)) were investigated in rat cardiac muscle. Skinned right ventricular trabeculae were isolated from wild-type (WT) and mutant homozygote (Ho) hearts expressing predominantly a smaller N2B isoform (2,970 kDa) and a giant N2BA-G isoform (3,830 kDa), respectively. Stretching WT and Ho trabeculae from SL 2.0 to 2.35 µm increased passive force, maximum Ca(2+)-activated force, and pCa(50), and it decreased n(H) and k(tr). Compared with WT trabeculae, the magnitude of SL-dependent changes in passive force, maximum Ca(2+)-activated force, pCa(50), and n(H) was significantly smaller in Ho trabeculae. These results suggests that, at least in rat ventricle, the magnitude of SL-dependent changes in passive force, maximum Ca(2+)-activated force, pCa(50), n(H), and k(tr) is defined by the titin isoform.

Cytoplasmic gamma-actin is not required for skeletal muscle development but its absence leads to a progressive myopathy.

Nonmuscle gamma(cyto)-actin is expressed at very low levels in skeletal muscle but uniquely localizes to costameres, the cytoskeletal networks that couple peripheral myofibrils to the sarcolemma. We generated and analyzed skeletal muscle-specific gamma(cyto)-actin knockout (Actg1-msKO) mice. Although muscle development proceeded normally, Actg1-msKO mice presented with overt muscle weakness accompanied by a progressive pattern of muscle fiber necrosis/regeneration. Functional deficits in whole-body tension and isometric twitch force were observed, consistent with defects in the connectivity between muscle fibers and/or myofibrils or at the myotendinous junctions. Surprisingly, gamma(cyto)-actin-deficient muscle did not demonstrate the fibrosis, inflammation, and membrane damage typical of several muscular dystrophies but rather presented with a novel progressive myopathy. Together, our data demonstrate an important role for minimally abundant but strategically localized gamma(cyto)-actin in adult skeletal muscle and describe a new mouse model to study the in vivo relevance of subcellular actin isoform sorting.

cMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium.

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development.

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca(2+) sensitivity of force (pCa(50)), PKA treatment has been shown to decrease pCa(50), presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca(2+)-independent force and maximum Ca(2+)-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I(1,1)/I(1,0)) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d(1,0)) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I(1,1)/I(1,0) and, as hypothesized, treatment with MLCK also increased I(1,1)/I(1,0), which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by 2 nm (3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca(2+) sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics.

The ventricles of small mammals express mostly alpha-myosin heavy chain (alpha-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express approximately 10% alpha-MHC on a predominately beta-MHC (slow isoform) background. In failing human ventricles, the amount of alpha-MHC is dramatically reduced, leading to the hypothesis that even small amounts of alpha-MHC on a predominately beta-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) for myosins composed of 100% alpha-MHC or beta-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% alpha-MHC or 100% beta-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentrations. The rate of ATP utilization was approximately 2.5-fold higher in preparations expressing 100% alpha-MHC compared with those expressing only beta-MHC, whereas k(tr) was 2-fold faster in the alpha-MHC myocardium. From these variables, we calculated f(app) to be approximately threefold higher for alpha-MHC than beta-MHC and g(app) to be twofold higher in alpha-MHC. Mathematical modeling of isometric twitches predicted that small increases in alpha-MHC significantly increased the rate of force development. These results suggest that low-level expression of alpha-MHC has significant effects on contraction kinetics.

Differential roles of cardiac myosin-binding protein C and cardiac troponin I in the myofibrillar force responses to protein kinase A phosphorylation.

The heart is remarkably adaptable in its ability to vary its function to meet the changing demands of the circulatory system. During times of physiological stress, cardiac output increases in response to increased sympathetic activity, which results in protein kinase A (PKA)-mediated phosphorylations of the myofilament proteins cardiac troponin (cTn)I and cardiac myosin-binding protein (cMyBP)-C. Despite the importance of this mechanism, little is known about the relative contributions of cTnI and cMyBP-C phosphorylation to increased cardiac contractility. Using engineered mouse lines either lacking cMyBP-C (cMyBP-C(-/-)) or expressing a non-PKA phosphorylatable cTnI (cTnI(ala2)), or both (cMyBP-C(-/-)/cTnI(ala2)), we investigated the roles of cTnI and cMyBP-C phosphorylation in the regulation of the stretch-activation response. PKA treatment of wild-type and cTnI(ala2) skinned ventricular myocardium accelerated stretch activation such that the response was indistinguishable from stretch activation of cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium; however, PKA had no effect on stretch activation in cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium. These results indicate that the acceleration of stretch activation in wild-type and cTnI(ala2) myocardium is caused by phosphorylation of cMyBP-C and not cTnI. We conclude that the primary effect of PKA phosphorylation of cTnI is reduced Ca(2+) sensitivity of force, whereas phosphorylation of cMyBP-C accelerates the kinetics of force development. These results predict that PKA phosphorylation of myofibrillar proteins in living myocardium contributes to accelerated relaxation in diastole and increased rates of force development in systole.

Recovery of left ventricular function following in vivo reexpression of cardiac myosin binding protein C.

The loss of cardiac myosin binding protein C (cMyBP-C) results in left ventricular dilation, cardiac hypertrophy, and impaired ventricular function in both constitutive and conditional cMyBP-C knockout (MYBPC3 null) mice. It remains unclear whether the structural and functional phenotypes expressed in the MYBPC3 null mouse are reversible, which is an important question, since reduced expression of cMyBP-C is an important cause of hypertrophic cardiomyopathy in humans. To investigate this question, we generated a cardiac-specific transgenic mouse model using a Tet-Off inducible system to permit the controlled expression of WT cMyBP-C on the MYBPC3 null background. Functional Tet-Off mice expressing WT cMyBP-C (FT-WT) were generated by crossing tetracycline transactivator mice with responder mice carrying the WT cMyBP-C transgene. Prior to dietary doxycycline administration, cMyBP-C was expressed at normal levels in FT-WT myocardium, which exhibited similar levels of steady-state force and in vivo left ventricular function as WT mice. Introduction of dietary doxycycline for four weeks resulted in a partial knockdown of cMyBP-C expression and commensurate impairment of systolic and diastolic function to levels approaching those observed in MYBPC 3 null mice. Subsequent withdrawal of doxycycline from the diet resulted in the reexpression of cMyBP-C to levels comparable to those observed in WT mice, along with near-complete recovery of in vivo ventricular function. These results show that the cardiac phenotypes associated with MYBPC3 null mice are reversible. Our work also validates the use of the Tet-Off inducible system as a means to study the mechanisms underlying hypertrophic cardiomyopathy.

Deletion of Enigma Homologue from the Z-disc slows tension development kinetics in mouse myocardium.

Enigma Homologue (ENH) is a component of the Z-disc, a structure that anchors actin filaments in the contractile unit of muscle, the sarcomere. Cardiac-specific ablation of ENH protein expression causes contractile dysfunction that ultimately culminates in dilated cardiomyopathy. However, whether ENH is involved in the regulation of myocardial contractility is unknown. To determine if ENH is required for the mechanical activity of cardiac muscle, we analyze muscle mechanics of isolated trabeculae from the hearts of ENH +/+ and ENH -/- mice. We detected no differences in steady-state mechanical properties but show that when muscle fibers are allowed to relax and then are restretched, the rate at which tension redevelops is depressed in ENH -/- mouse myocardium relative to that in ENH +/+ myocardium. SDS-PAGE analysis demonstrated that the expression of ß-myosin heavy chain is increased in ENH -/- mouse myocardium, which could partially, but not completely, account for the depression in tension redevelopment kinetics. Using top-down proteomics analysis, we found that the expression of other thin/thick filament regulatory proteins is unaltered, although the phosphorylation of a cardiac troponin T isoform, cardiac troponin I, and myosin regulatory light chain is decreased in ENH -/- mouse myocardium. Nevertheless, these alterations are very small and thus insufficient to explain slowed tension redevelopment kinetics in ENH -/- mouse myocardium. These data suggest that the ENH protein influences tension redevelopment kinetics in mouse myocardium, possibly by affecting cross-bridge cycling kinetics. Previous studies also indicate that ablation of specific Z-disc proteins in myocardium slows contraction kinetics, which could also be a contributing factor in this study.

Transmural variation in myosin heavy chain isoform expression modulates the timing of myocardial force generation in porcine left ventricle.

Recent studies have shown that the sequence and timing of mechanical activation of myocardium vary across the ventricular wall. However, the contributions of variable expression of myofilament protein isoforms in mediating the timing of myocardial activation in ventricular systole are not well understood. To assess the functional consequences of transmural differences in myofilament protein expression, we studied the dynamic mechanical properties of multicellular skinned preparations isolated from the sub-endocardial and sub-epicardial regions of the porcine ventricular midwall. Compared to endocardial fibres, epicardial fibres exhibited significantly faster rates of stretch activation and force redevelopment (k(tr)), although the amount of force produced at a given [Ca2+] was not significantly different. Consistent with these results, SDS-PAGE analysis revealed significantly elevated expression of alpha myosin heavy chain (MHC) isoform in epicardial fibres (13 +/- 1%) versus endocardial fibres (3 +/- 1%). Linear regression analysis revealed that the apparent rates of delayed force development and force decay following stretch correlated with MHC isoform expression (r2 = 0.80 and r2 = 0.73, respectively, P < 0.05). No differences in the relative abundance or phosphorylation status of other myofilament proteins were detected. These data show that transmural differences in MHC isoform expression contribute to regional differences in dynamic mechanical function of porcine left ventricles, which in turn modulate the timing of force generation across the ventricular wall and work production during systole.

Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocardium is eliminated by ablation of cMyBP-C.

Beta-adrenergic agonists induce protein kinase A (PKA) phosphorylation of the cardiac myofilament proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI), resulting in enhanced systolic function, but the relative contributions of cMyBP-C and cTnI to augmented contractility are not known. To investigate possible roles of cMyBP-C in this response, we examined the effects of PKA treatment on the rate of force redevelopment and the stretch activation response in skinned ventricular myocardium from both wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) myocardium. In WT myocardium, PKA treatment accelerated the rate of force redevelopment and the stretch activation response, resulting in a shorter time to the peak of delayed force development when the muscle was stretched to a new isometric length. Ablation of cMyBP-C accelerated the rate of force redevelopment and stretch activation response to a degree similar to that observed in PKA treatment of WT myocardium; however, PKA treatment had no effect on the rate of force development and the stretch activation response in null myocardium. These results indicate that ablation of cMyBP-C and PKA treatment of WT myocardium have similar effects on cross-bridge cycling kinetics and suggest that PKA phosphorylation of cMyBP-C accelerates the rate of force generation and thereby contributes to the accelerated twitch kinetics observed in living myocardium during beta-adrenergic stimulation.

Organ-level right ventricular dysfunction with preserved Frank-Starling mechanism in a mouse model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rapidly fatal disease in which mortality is due to right ventricular (RV) failure. It is unclear whether RV dysfunction initiates at the organ level or the subcellular level or both. We hypothesized that chronic pressure overload-induced RV dysfunction begins at the organ level with preserved Frank-Starling mechanism in myocytes. To test this hypothesis, we induced PAH with Sugen + hypoxia (HySu) in mice and measured RV whole organ and subcellular functional changes by in vivo pressure-volume measurements and in vitro trabeculae length-tension measurements, respectively, at multiple time points for up to 56 days. We observed progressive changes in RV function at the organ level: in contrast to early PAH (14-day HySu), in late PAH (56-day HySu) ejection fraction and ventricular-vascular coupling were decreased. At the subcellular level, direct measurements of myofilament contraction showed that RV contractile force was similarly increased at any stage of PAH development. Moreover, cross-bridge kinetics were not changed and length dependence of force development (Frank-Starling relation) were not different from baseline in any PAH group. Histological examinations confirmed increased cardiomyocyte cross-sectional area and decreased von Willebrand factor expression in RVs with PAH. In summary, RV dysfunction developed at the organ level with preserved Frank-Starling mechanism in myofilaments, and these results provide novel insight into the development of RV dysfunction, which is critical to understanding the mechanisms of RV failure. NEW & NOTEWORTHY A multiscale investigation of pulmonary artery pressure overload in mice showed time-dependent organ-level right ventricular (RV) dysfunction with preserved Frank-Starling relations in myofilaments. Our findings provide novel insight into the development of RV dysfunction, which is critical to understanding mechanisms of RV failure.

Acceleration of stretch activation in murine myocardium due to phosphorylation of myosin regulatory light chain.

The regulatory light chains (RLCs) of vertebrate muscle myosins bind to the neck region of the heavy chain domain and are thought to play important structural roles in force transmission between the cross-bridge head and thick filament backbone. In vertebrate striated muscles, the RLCs are reversibly phosphorylated by a specific myosin light chain kinase (MLCK), and while phosphorylation has been shown to accelerate the kinetics of force development in skeletal muscle, the effects of RLC phosphorylation in cardiac muscle are not well understood. Here, we assessed the effects of RLC phosphorylation on force, and the kinetics of force development in myocardium was isolated in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC, subsequently skinned, and then treated with MLCK to phosphorylate RLC. Since RLC phosphorylation may be an important determinant of stretch activation in myocardium, we recorded the force responses of skinned myocardium to sudden stretches of 1% of muscle length both before and after treatment with MLCK. MLCK increased RLC phosphorylation, increased the Ca(2+) sensitivity of isometric force, reduced the steepness of the force-pCa relationship, and increased both Ca(2+)-activated and Ca(2+)-independent force. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force, i.e., stretch activation, to levels greater than pre-stretch force. MLCK had profound effects on the stretch activation responses during maximal and submaximal activations: the amplitude and rate of force decay after stretch were significantly reduced, and the rate of delayed force recovery was accelerated and its amplitude reduced. These data show that RLC phosphorylation increases force and the rate of cross-bridge recruitment in murine myocardium, which would increase power generation in vivo and thereby enhance systolic function.

Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle.

Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficient mdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments.

Altered Right Ventricular Mechanical Properties Are Afterload Dependent in a Rodent Model of Bronchopulmonary Dysplasia.

Infants born premature are at increased risk for development of bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), and ultimately right ventricular (RV) dysfunction, which together carry a high risk of neonatal mortality. However, the role alveolar simplification and abnormal pulmonary microvascular development in BPD affects RV contractile properties is unknown. We used a rat model of BPD to examine the effect of hyperoxia-induced PH on RV contractile properties. We measured in vivo RV pressure as well as passive force, maximum Ca2+ activated force, calcium sensitivity of force (pCa50) and rate of force redevelopment (ktr) in RV skinned trabeculae isolated from hearts of 21-and 35-day old rats pre-exposed to 21% oxygen (normoxia) or 85% oxygen (hyperoxia) for 14 days after birth. Systolic and diastolic RV pressure were significantly higher at day 21 in hyperoxia exposed rats compared to normoxia control rats, but normalized by 35 days of age. Passive force, maximum Ca2+ activated force, and calcium sensitivity of force were elevated and cross-bridge cycling kinetics depressed in 21-day old hyperoxic trabeculae, whereas no differences between normoxic and hyperoxic trabeculae were seen at 35 days. Myofibrillar protein analysis revealed that 21-day old hyperoxic trabeculae had increased levels of beta-myosin heavy chain (ß-MHC), atrial myosin light chain 1 (aMLC1; often referred to as essential light chain), and slow skeletal troponin I (ssTnI) compared to age matched normoxic trabeculae. On the other hand, 35-day old normoxic and hyperoxic trabeculae expressed similar level of α- and ß-MHC, ventricular MLC1 and predominantly cTnI. These results suggest that neonatal exposure to hyperoxia increases RV afterload and affect both the steady state and dynamic contractile properties of the RV, likely as a result of hyperoxia-induced expression of ß-MHC, delayed transition of slow skeletal TnI to cardiac TnI, and expression of atrial MLC1. These hyperoxia-induced changes in contractile properties are reversible and accompany the resolution of PH with further developmental age, underscoring the importance of reducing RV afterload to allow for normalization of RV function in both animal models and humans with BPD.

Ablation of the cardiac-specific gene leucine-rich repeat containing 10 (Lrrc10) results in dilated cardiomyopathy.

Leucine-rich repeat containing 10 (LRRC10) is a cardiac-specific protein exclusively expressed in embryonic and adult cardiomyocytes. However, the role of LRRC10 in mammalian cardiac physiology remains unknown. To determine if LRRC10 is critical for cardiac function, Lrrc10-null (Lrrc10(-/-)) mice were analyzed. Lrrc10(-) (/-) mice exhibit prenatal systolic dysfunction and dilated cardiomyopathy in postnatal life. Importantly, Lrrc10(-/-) mice have diminished cardiac performance in utero, prior to ventricular dilation observed in young adults. We demonstrate that LRRC10 endogenously interacts with α-actinin and α-actin in the heart and all actin isoforms in vitro. Gene expression profiling of embryonic Lrrc10(-/-) hearts identified pathways and transcripts involved in regulation of the actin cytoskeleton to be significantly upregulated, implicating dysregulation of the actin cytoskeleton as an early defective molecular signal in the absence of LRRC10. In contrast, microarray analyses of adult Lrrc10(-/-) hearts identified upregulation of oxidative phosphorylation and cardiac muscle contraction pathways during the progression of dilated cardiomyopathy. Analyses of hypertrophic signal transduction pathways indicate increased active forms of Akt and PKCε in adult Lrrc10(-/-) hearts. Taken together, our data demonstrate that LRRC10 is essential for proper mammalian cardiac function. We identify Lrrc10 as a novel dilated cardiomyopathy candidate gene and the Lrrc10(-/-) mouse model as a unique system to investigate pediatric cardiomyopathy.

Protein kinase A-induced myofilament desensitization to Ca(2+) as a result of phosphorylation of cardiac myosin-binding protein C.

In skinned myocardium, cyclic AMP-dependent protein kinase A (PKA)-catalyzed phosphorylation of cardiac myosin-binding protein C (cMyBP-C) and cardiac troponin I (cTnI) is associated with a reduction in the Ca(2+) responsiveness of myofilaments and an acceleration in the kinetics of cross-bridge cycling, although the respective contribution of these two proteins remains controversial. To further examine the relative roles that cTnI and cMyBP-C phosphorylation play in altering myocardial function, we determined the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in control and PKA-treated mouse myocardium (isolated in the presence of 2,3-butanedione monoxime) expressing: (a) phosphorylatable cTnI and cMyBP-C (wild type [WT]), (b) phosphorylatable cTnI on a cMyBP-C-null background (cMyBP-C(-/-)), (c) nonphosphorylatable cTnI with serines(23/24/43/45) and threonine(144) mutated to alanines (cTnI(Ala5)), and (d) nonphosphorylatable cTnI on a cMyBP-C-null background (cTnI(Ala5)/cMyBP-C(-/-)). Here, PKA treatment decreased pCa(50) in WT, cTnI(Ala5), and cMyBP-C(-/-) myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in cTnI(Ala5)/cMyBP-C(-/-) myocardium. In WT and cTnI(Ala5) myocardium, PKA treatment also increased k(tr) at submaximal levels of activation; however, PKA treatment did not have an effect on k(tr) in cMyBP-C(-/-) or cTnI(Ala5)/cMyBP-C(-/-) myocardium. In addition, reconstitution of cTnI(Ala5)/cMyBP-C(-/-) myocardium with recombinant cMyBP-C restored the effects of PKA treatment on pCa(50) and k(tr) reported in cTnI(Ala5) myocardium. Collectively, these results indicate that the attenuation in myofilament force response to PKA occurs as a result of both cTnI and cMyBP-C phosphorylation, and that the reduction in pCa(50) mediated by cMyBP-C phosphorylation most likely arises from an accelerated cross-bridge cycling kinetics partly as a result of an increased rate constant of cross-bridge detachment.

Cytoplasmic gamma-actin contributes to a compensatory remodeling response in dystrophin-deficient muscle.

Dystrophin mechanically links the costameric cytoskeleton and sarcolemma, yet dystrophin-deficient muscle exhibits abnormalities in cell signaling, gene expression, and contractile function that are not clearly understood. We generated new antibodies specific for cytoplasmic gamma-actin and confirmed that gamma-actin most predominantly localized to the sarcolemma and in a faint reticular lattice within normal muscle cells. However, we observed that gamma-actin levels were increased 10-fold at the sarcolemma and within the cytoplasm of striated muscle cells from dystrophin-deficient mdx mice. Transgenic overexpression of the dystrophin homologue utrophin, or functional dystrophin constructs in mdx muscle, restored gamma-actin to normal levels, whereas gamma-actin remained elevated in mdx muscle expressing nonfunctional dystrophin constructs. We conclude that increased cytoplasmic gamma-actin in dystrophin-deficient muscle may be a compensatory response to fortify the weakened costameric lattice through recruitment of parallel mechanical linkages. However, the presence of excessive myoplasmic gamma-actin may also contribute to altered cell signaling or gene expression in dystrophin-deficient muscle.