Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes.

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Cardiac contractile dysfunction and protein kinase C-mediated myofilament phosphorylation in disease and aging.

Increases in protein kinase C (PKC) are associated with diminished cardiac function, but the contribution of downstream myofilament phosphorylation is debated in human and animal models of heart failure. The current experiments evaluated PKC isoform expression, downstream cardiac troponin I (cTnI) S44 phosphorylation (p-S44), and contractile function in failing (F) human myocardium, and in rat models of cardiac dysfunction caused by pressure overload and aging. In F human myocardium, elevated PKCα expression and cTnI p-S44 developed before ventricular assist device implantation. Circulatory support partially reduced PKCα expression and cTnI p-S44 levels and improved cellular contractile function. Gene transfer of dominant negative PKCα (PKCαDN) into F human myocytes also improved contractile function and reduced cTnI p-S44. Heightened cTnI phosphorylation of the analogous residue accompanied reduced myocyte contractile function in a rat model of pressure overload and in aged Fischer 344 × Brown Norway F1 rats (≥26 mo). Together, these results indicate PKC-targeted cTnI p-S44 accompanies cardiac cellular dysfunction in human and animal models. Interfering with PKCα activity reduces downstream cTnI p-S44 levels and partially restores function, suggesting cTnI p-S44 may be a useful target to improve contractile function in the future.

Troponin I modulation of cardiac performance: Plasticity in the survival switch.

Signaling complexes targeting the myofilament are essential in modulating cardiac performance. A central target of this signaling is cardiac troponin I (cTnI) phosphorylation. This review focuses on cTnI phosphorylation as a model for myofilament signaling, discussing key gaps and future directions towards understanding complex myofilament modulation of cardiac performance. Human heart cTnI is phosphorylated at 14 sites, giving rise to a complex modulatory network of varied functional responses. For example, while classical Ser23/24 phosphorylation mediates accelerated relaxation, protein kinase C phosphorylation of cTnI serves as a brake on contractile function. Additionally, the functional response of cTnI multi-site phosphorylation cannot necessarily be predicted from the response of individual sites alone. These complexities underscore the need for systematically evaluating single and multi-site phosphorylation on myofilament cellular and in vivo contractile function. Ultimately, a complete understanding of these multi-site responses requires work to establish site occupancy and dominance, kinase/phosphatase signaling balance, and the function of adaptive secondary phosphorylation. As cTnI phosphorylation is essential for modulating cardiac performance, future insight into the complex role of cTnI phosphorylation is important to establish sarcomere signaling in the healthy heart as well as identification of novel myofilament targets in the treatment of disease.

Secondary phosphorylation in myocytes expressing FLAG-tagged and non-tagged phospho-mimetic cardiac troponin I.

Secondary phosphorylation develops in myocytes expressing phospho-mimetic cardiac troponin I (cTnI) but it is not known whether multiple substitutions (e.g. cTnISDTD and cTnIS4D) cause preferential phosphorylation of the remaining endogenous or the phospho-mimetic cTnI in intact myocytes. Western analysis was performed to determine whether the FLAG/total cTnI ratios are similar for phosphorylated versus total cTnI in myocytes expressing phospho-mimetic cTnI with Asp(D) substitutions at S43/45 plus S23/24 (cTnIS4D) or T144 (cTnISDTD). Representative Western analysis of phosphorylated S23/24 (p-S23/24) and S150 (p-S150) are presented along with re-probes using an antibody which detects all cTnI (MAB1691 Ab). The level of p-S150 also is compared to results obtained using single S43D and/or S45D phospho-mimetic substitutions. These results are discussed in more detail in Lang et al. [1].

Functional communication between PKC-targeted cardiac troponin I phosphorylation sites.

Increased protein kinase C (PKC) activity is associated with heart failure, and can target multiple cardiac troponin I (cTnI) residues in myocytes, including S23/24, S43/45 and T144. In earlier studies, cTnI-S43D and/or -S45D augmented S23/24 and T144 phosphorylation, which suggested there is communication between clusters. This communication is now explored by evaluating the impact of phospho-mimetic cTnI S43/45D combined with S23/24D (cTnIS4D) or T144D (cTnISDTD). Gene transfer of epitope-tagged cTnIS4D and cTnISDTD into adult cardiac myocytes progressively replaced endogenous cTnI. Partial replacement with cTnISDTD or cTnIS4D accelerated the time to peak (TTP) shortening and time to 50% re-lengthening (TTR50%) on day 2, but peak shortening was only diminished by cTnIS4D. Extensive cTnIS4D replacement continued to accelerate TTP, and decrease shortening amplitude, while TTR50% returned to baseline levels on day 4. In contrast, cTnISDTD modestly reduced shortening amplitude and continued to accelerate myocyte TTP and TTR50%. These results indicate cTnIS43/45 communicates with S23/24 and T144, with S23/24 exacerbating and T144 attenuating the S43/45D-dependent functional deficit. In addition, more severe functional alterations in cTnIS4D myocytes were accompanied by higher levels of secondary phosphorylation compared to cTnISDTD. These results suggest that secondary phosphorylation helps to maintain steady-state contractile function during chronic cTnI phosphorylation at PKC sites.

Contribution of Post-translational Phosphorylation to Sarcomere-Linked Cardiomyopathy Phenotypes.

Secondary shifts develop in post-translational phosphorylation of sarcomeric proteins in multiple animal models of inherited cardiomyopathy. These signaling alterations together with the primary mutation are predicted to contribute to the overall cardiac phenotype. As a result, identification and integration of post-translational myofilament signaling responses are identified as priorities for gaining insights into sarcomeric cardiomyopathies. However, significant questions remain about the nature and contribution of post-translational phosphorylation to structural remodeling and cardiac dysfunction in animal models and human patients. This perspective essay discusses specific goals for filling critical gaps about post-translational signaling in response to these inherited mutations, especially within sarcomeric proteins. The discussion focuses primarily on pre-clinical analysis of animal models and defines challenges and future directions in this field.

Functionally conservative substitutions at cardiac troponin I S43/45.

A phospho-null Ala substitution at protein kinase C (PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca(2+) transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance.

Differential protein expression and basal lamina remodeling in human heart failure.

PURPOSE: A goal of this study was to identify and investigate previously unrecognized components of the remodeling process in the progression to heart failure by comparing protein expression in ischemic failing (F) and nonfailing (NF) human hearts. EXPERIMENTAL DESIGN: Protein expression differences were investigated using multidimensional protein identification and validated by Western analysis. This approach detected basal lamina (BL) remodeling, and further studies analyzed samples for evidence of structural BL remodeling. A rat model of pressure overload (PO) was studied to determine whether nonischemic stressors also produce BL remodeling and impact cellular adhesion. RESULTS: Differential protein expression of collagen IV, laminin α2, and nidogen-1 indicated BL remodeling develops in F versus NF hearts Periodic disruption of cardiac myocyte BL accompanied this process in F, but not NF heart. The rat PO myocardium also developed BL remodeling and compromised myocyte adhesion compared to sham controls. CONCLUSIONS AND CLINICAL RELEVANCE: Differential protein expression and evidence of structural and functional BL alterations develop during heart failure. The compromised adhesion associated with this remodeling indicates a high potential for dysfunctional cellular integrity and tethering in failing myocytes. Therapeutically targeting BL remodeling could slow or prevent the progression of heart disease.

Gene transfer into cardiac myocytes.

Traditional methods for DNA transfection are often inefficient and toxic for terminally differentiated cells, such as cardiac myocytes. Vector-based gene transfer is an efficient approach for introducing exogenous cDNA into these types of primary cell cultures. In this chapter, separate protocols for adult rat cardiac myocyte isolation and gene transfer with recombinant adenovirus are provided and are routinely utilized for studying the effects of sarcomeric proteins on myofilament function.

Independent modulation of contractile performance by cardiac troponin I Ser43 and Ser45 in the dynamic sarcomere.

Protein kinase C (PKC) targets cardiac troponin I (cTnI) S43/45 for phosphorylation in addition to other residues. During heart failure, cTnI S43/45 phosphorylation is elevated, and yet there is ongoing debate about its functional role due, in part, to the emergence of complex phenotypes in animal models. The individual functional influences of phosphorylated S43 and S45 also are not yet known. The present study utilizes viral gene transfer of cTnI with phosphomimetic S43D and/or S45D substitutions to evaluate their individual and combined influences on function in intact adult cardiac myocytes. Partial replacement (≤40%) with either cTnIS43D or cTnIS45D reduced the amplitude of contraction, and cTnIS45D slowed contraction and relaxation rates, while there were no significant changes in function with cTnIS43/45D. More extensive replacement (≥70%) with cTnIS43D, cTnIS45D, and cTnIS43/45D each reduced the amplitude of contraction. Additional experiments also showed cTnIS45D reduced myofilament Ca(2+) sensitivity of tension. At the same time, shortening rates returned toward control values with cTnIS45D and the later stages of relaxation also became accelerated in myocytes expressing cTnIS43D and/or S45D. Further studies demonstrated this behavior coincided with adaptive changes in myofilament protein phosphorylation. Taken together, the results observed in myocytes expressing cTnIS43D and/or S45D suggest these 2 residues reduce function via independent mechanism(s). The changes in function associated with the onset of adaptive myofilament signaling suggest the sarcomere is capable of fine tuning PKC-mediated cTnIS43/45 phosphorylation and contractile performance. This modulatory behavior also provides insight into divergent phenotypes reported in animal models with cTnI S43/45 phosphomimetic substitutions.

Sarcomere mutation-specific expression patterns in human hypertrophic cardiomyopathy.

BACKGROUND: Heterozygous mutations in sarcomere genes in hypertrophic cardiomyopathy (HCM) are proposed to exert their effect through gain of function for missense mutations or loss of function for truncating mutations. However, allelic expression from individual mutations has not been sufficiently characterized to support this exclusive distinction in human HCM. METHODS AND RESULTS: Sarcomere transcript and protein levels were analyzed in septal myectomy and transplant specimens from 46 genotyped HCM patients with or without sarcomere gene mutations and 10 control hearts. For truncating mutations in MYBPC3, the average ratio of mutant:wild-type transcripts was ≈1:5, in contrast to ≈1:1 for all sarcomere missense mutations, confirming that nonsense transcripts are uniquely unstable. However, total MYBPC3 mRNA was significantly increased by 9-fold in HCM samples with MYBPC3 mutations compared with control hearts and with HCM samples without sarcomere gene mutations. Full-length MYBPC3 protein content was not different between MYBPC3 mutant HCM and control samples, and no truncated proteins were detected. By absolute quantification of abundance with multiple reaction monitoring, stoichiometric ratios of mutant sarcomere proteins relative to wild type were strikingly variable in a mutation-specific manner, with the fraction of mutant protein ranging from 30% to 84%. CONCLUSIONS: These results challenge the concept that haploinsufficiency is a unifying mechanism for HCM caused by MYBPC3 truncating mutations. The range of allelic imbalance for several missense sarcomere mutations suggests that certain mutant proteins may be more or less stable or incorporate more or less efficiently into the sarcomere than wild-type proteins. These mutation-specific properties may distinctly influence disease phenotypes.

Myocardial infarction-induced N-terminal fragment of cardiac myosin-binding protein C (cMyBP-C) impairs myofilament function in human myocardium.

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca(2+) transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 µm) and long (2.3 µm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca(2+) sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca(2+) sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.

Agonist activated PKCßII translocation and modulation of cardiac myocyte contractile function.

Elevated protein kinase C ßII (PKCßII) expression develops during heart failure and yet the role of this isoform in modulating contractile function remains controversial. The present study examines the impact of agonist-induced PKCßII activation on contractile function in adult cardiac myocytes. Diminished contractile function develops in response to low dose phenylephrine (PHE, 100 nM) in controls, while function is preserved in response to PHE in PKCßII-expressing myocytes. PHE also caused PKCßII translocation and a punctate distribution pattern in myocytes expressing this isoform. The preserved contractile function and translocation responses to PHE are blocked by the inhibitor, LY379196 (30 nM) in PKCßII-expressing myocytes. Further analysis showed downstream protein kinase D (PKD) phosphorylation and phosphatase activation are associated with the LY379196-sensitive contractile response. PHE also triggered a complex pattern of end-target phosphorylation in PKCßII-expressing myocytes. These patterns are consistent with bifurcated activation of downstream signaling activity by PKCßII.

Myofilament incorporation and contractile function after gene transfer of cardiac troponin I Ser43/45Ala.

Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70-80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca(2+) sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca(2+) transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.

Substrate stiffness affects sarcomere and costamere structure and electrophysiological function of isolated adult cardiomyocytes.

INTRODUCTION: The mechanical environment is a key regulator of function in cardiomyocytes. We studied the role of substrate stiffness on the organization of sarcomeres and costameres in adult rat cardiomyocytes and further examined the resulting changes in cell shortening and calcium dynamics. METHODS: Cardiomyocytes isolated from adult rats were plated on laminin-coated polydimethylsiloxane substrates of defined stiffness (255 kPa, 117 kPa, 27 kPa, and 7 kPa) for 48 h. Levels of α-actinin and ß1 integrins were determined by immunofluoresence imaging and immunoblotting, both in the absence and presence of the phosphatase inhibitor calyculin A. Quantitative reverse transcriptase polymerase chain reaction was used to measure message levels of key structural proteins (α-actinin, α7 integrin, ß1 integrin, vinculin). Sarcomere shortening and calcium dynamics were measured at 2, 24, and 48 h. RESULTS: Overall cardiomyocyte morphology was similar on all substrates. However, well organized sarcomere structures were observed on only the stiffest (255 kPa) and the most compliant (7 kPa) substrates. Levels of α-actinin in cells were the same on all substrates, while message levels of structural proteins were up-regulated on substrates of intermediate stiffness. Inhibition of phosphatase activity blocked the degradation of contractile structures, but altered overall cardiomyocyte morphology. Shortening and calcium dynamics also were dependent on substrate stiffness; however, there was no clear causative relationship between the phenomena. CONCLUSIONS: Extracellular matrix stiffness can affect structural remodeling by adult cardiomyocytes, and the resulting contractile activity. These findings illuminate changes in cardiomyocyte function in cardiac fibrosis, and may suggest cardiac-specific phosphatases as a target for therapeutic intervention.

Structure-activity studies of RFamide-related peptide-1 identify a functional receptor antagonist and novel cardiac myocyte signaling pathway involved in contractile performance.

Human RFamide-related peptide-1 (hRFRP-1, MPHSFANLPLRF-NH(2)) binds to neuropeptide FF receptor 2 (NPFF(2)R) to dramatically diminish cardiovascular performance. hRFRP-1 and its signaling pathway may provide targets to address cardiac dysfunction. Here, structure-activity relationship, transcript, Ca(2+) transient, and phospholabeling data indicate the presence of a hRFRP-1 pathway in cardiomyocytes. Alanyl-substituted and N-terminal truncated analogues identified that R(11) was essential for activity, hRFRP-1((8-12)) mimicked hRFRP-1, and [A(11)]hRFRP-1((8-12)) antagonized the effect of hRFRP-1 in cellular and integrated cardiac performance. RFRP and NPFF(2)R transcripts were amplified from cardiomyocytes and heart. Maintenance of the Ca(2+) transient when hRFRP-1 impaired myocyte shortening indicated the myofilament was its primary downstream target. Enhanced myofilament protein phosphorylation detected after hRFRP-1 treatment but absent in [A(11)]hRFRP-1((8-12))-treated cells was consistent with this result. Protein kinase C (PKC) but not PKA inhibitor diminished the influence of hRFRP-1 on the Ca(2+) transient. Molecules targeting this pathway may help address cardiovascular disease.

PKCßII modulation of myocyte contractile performance.

Significant up-regulation of the protein kinase Cß(II) (PKCß(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCß(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCß(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCß(II) was distributed in a peri-nuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCß(II) (PKCßDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCß(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCß(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCß inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCß(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCß(II) increased Ca(2+)-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCß(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes.

Reduced serum content and increased matrix stiffness promote the cardiac myofibroblast transition in 3D collagen matrices.

INTRODUCTION: The fibroblast-myofibroblast transition is an important event in the development of cardiac fibrosis and scar formation initiated after myocardial ischemia. The goals of the present study were to better understand the contribution of environmental factors to this transition and determine whether myofibroblasts provide equally important feedback to the surrounding environment. METHODS: The influence of matrix stiffness and serum concentration on the myofibroblast transition was assessed by measuring message levels of a panel of cardiac fibroblast phenotype markers using quantitative reverse transcriptase polymerase chain reaction. Cell-mediated gel compaction measured the influence of environmental factors on cardiac fibroblast contractility. Immunohistochemistry characterized alpha-smooth muscle actin expression and cell morphology, while static and dynamic compression testing evaluated the effect of the cell response on the mechanical properties of the cell-seeded collagen hydrogels. RESULTS: Both reduced serum content and increased matrix stiffness contributed to the myofibroblast transition, as indicated by contractile compaction of the gels, increased message levels of col3α1 and alpha-smooth muscle actin, and a less stellate morphology. However, the effects of serum and matrix stiffness were not additive. Mechanical testing indicated that reduced serum content increased the initial elastic modulus of cell-seeded gels and that gels lost their viscous character with time. CONCLUSIONS: The results suggest that reduced serum and increased matrix stiffness promote the myofibroblast phenotype in the myocardium. This transition both enhances and is promoted by matrix stiffness, indicating the presence of positive feedback that may contribute to the pathogenesis of cardiac fibrosis.

Burn-induced heart failure: lipopolysaccharide binding protein improves burn and endotoxin-induced cardiac contractility deficits.

BACKGROUND: Burn injury is frequently complicated by bacterial infection. Following burn injury, exposure to endotoxin produces a measurable decrease in cardiomyocyte sarcomere contractile function. Lipopolysaccharide-binding protein (LBP) is an acute phase protein that potentiates the recognition of lipopolysaccharide (LPS) by binding to the lipid A moiety of LPS. In this study, we sought to determine the effect of recombinant rat LBP (rLBP) on cardiomyocyte sarcomere function after burn or sham injury in the presence or absence of bacterial endotoxin. METHODS: Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 24 h post-injury, cardiomyocytes were isolated, plated at 50,000 cells/well, and incubated with 50 µg/mL LPS and rLBP or chloramphenicol acetyltransferase (BVCat, an irrelevant control protein produced using the same expression system as rLBP) at concentrations by volume of 1%, 5%, 10%, and 30%. Subsets of cardiomyocytes were incubated with 5% rat serum or 30% rLBP and blocking experiments were conducted using an LBP-like synthetic peptide (LBPK95A). In vitro sarcomere function was measured using a variable rate video camera system with length detection software. RESULTS: Co-culture of burn and sham injury derived cardiomyocytes with high-dose rLBP in the presence of LPS resulted in a significant reduction to the functional impairment observed in peak sarcomere shortening following exposure to LPS alone. LBP-like peptide LBPK95A at a concentration of 20 µg/mL, in the presence of LPS, abolished the ability of 30% rLBP and 5% rat serum to restore peak sarcomere shortening of cardiomyocytes isolated following burn injury to levels of function exhibited in the absence of endotoxin exposure. CONCLUSIONS: In the setting of LPS challenge following burn injury, rLBP at high concentrations restores cardiomyocyte sarcomere contractile function in vitro. Rather than potentiating the recognition of LPS by the cellular LPS receptor complex, rLBP at high concentrations likely results in an inhibitory binding effect that minimizes the impact of endotoxin exposure on cardiomyocyte function following thermal injury.