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Contractile dysfunction and Ca 2+ 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 Ca 2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca 2+ 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 Ca 2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

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., (© 2019 Ravichandran et al.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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].

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 (TTR 50% ) on day 2, but peak shortening was only diminished by cTnIS4D. Extensive cTnIS4D replacement continued to accelerate TTP, and decrease shortening amplitude, while TTR 50% returned to baseline levels on day 4. In contrast, cTnISDTD modestly reduced shortening amplitude and continued to accelerate myocyte TTP and TTR 50% . 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

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., (© 2014 American Heart Association, Inc.)

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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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 β1 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, β1 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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Elevated protein kinase C βII (PKCβII) 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 PKCβII 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 PKCβII-expressing myocytes. PHE also caused PKCβII 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 PKCβII-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 PKCβII-expressing myocytes. These patterns are consistent with bifurcated activation of downstream signaling activity by PKCβII.

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.

Significant up-regulation of the protein kinase Cβ(II) (PKCβ(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCβ(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCβ(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCβ(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 PKCβ(II) (PKCβDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCβ(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCβ(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCβ inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCβ(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCβ(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 PKCβ(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cardiac fibroblasts are exposed to both cyclic strain and interstitial fluid flow in the myocardium. The balance of these stimuli is affected by fibrotic scarring, during which the fibroblasts transition to a myofibroblast phenotype. The present study investigates the mechanisms by which cardiac fibroblasts seeded in three-dimensional (3D) collagen gels differentiate between strain and fluid flow. Neonatal cardiac fibroblast-seeded 3D collagen gels were exposed to interstitial flow and/or cyclic strain and message levels of collagens type I and III, transforming growth factor β1 (TGF-β1), and α-smooth muscle actin (α-SMA) were assessed. Flow was found to significantly increase and strain to decrease expression of myofibroblast markers. Corresponding immunofluorescence indicated that flow and strain differentially regulated α-SMA protein expression. The effect of flow was inhibited by exposure to losartan, an angiotensin II type 1 receptor (AT1R) blocker, and by introduction of shRNA constructs limiting AT1R expression. Blocking of TGF-β also inhibited the myofibroblast transition, suggesting that flow-mediated cell signaling involved both AT1R and TGF-β1. Reduced smad2 phosphorylation in response to cyclic strain suggested that TGF-β is part of the mechanism by which cardiac fibroblasts differentiate between strain-induced and flow-induced mechanical stress. Our experiments show that fluid flow and mechanical deformation have distinct effects on cardiac fibroblast phenotype. Our data suggest a mechanism in which fluid flow directly acts on AT1R and causes increased TGF-β1 expression, whereas cyclic strain reduces activation of smad proteins. These results have relevance to the pathogenesis and treatment of heart failure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Peptides influence cardiac dysfunction; however, peptidergic modulation of contractile performance remains relatively uncharacterized. We identified a novel human peptide that modulates mammalian contractile performance. Members of the FMRFamide-related peptide (FaRP) family contain a C-terminal RFamide but structurally variant N-terminal extension. We report human RFamide-related peptide-1 (hRFRP-1) and rat RFRP-1 rapidly and reversibly decreased shortening and relaxation in isolated mammalian cardiac myocytes in a dose dependent manner. The mammalian FaRP, 26RFa, structurally related to RFRP-1 by only an RFamide did not influence myocyte contractile function. The protein kinase C (PKC) inhibitor bisindolylmaleimide-1 blocked hRFRP-1 activity. Pretreatment with pertussis toxin (PTX) did not diminish hRFRP-1 influence on contractile function. In addition, intravenous injection of hRFRP-1 in mice decreased heart rate, stroke volume, ejection fraction, and cardiac output. Collectively these findings are consistent with the conclusion RFRP-1 is an endogenous signaling molecule that activates PKC and acts through a PTX-insensitive pathway to modulate cardiac contractile function. Taken together these negative chronotropic, inotropic, and lusitropic effects of hRFRP-1 are significant; they suggest direct acute cellular and organ-level responses in mammalian heart. This is the first known study to identify a mammalian FaRP with cardio-depressant effects, opening a new area of research on peptidergic modulation of contractile performance. The high degree of RFRP structure conservation from amphibians to mammals, and similarity to invertebrate cardioinhibitory peptides suggests RFRP-1 is involved in important physiological functions. Elucidation of mechanisms involved in hRFRP-1 synthesis, release, and signaling may aid the development of strategies to prevent or attenuate cardiac dysfunction., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Topical inhibition of activated p38 MAPK within burn wounds attenuates the local and systemic inflammatory response. In this study, we investigated the effects of local activated p38 MAPK inhibition on burn-induced cardiac dysfunction., Methods: Using a standardized rat model of scald burn injury, rats were given a 30% total body surface area partial thickness burn or sham injury, and the wounds were treated with an activated p38 MAPK inhibitor (SB) or vehicle. Systemic blood pressure measurements were recorded in vivo followed by in vitro assessment of sarcomere contraction in single-cell suspensions of isolated cardiomyocytes., Results: Systolic blood pressure or maximum left ventricular pressures in vivo and peak cardiomyocyte sarcomere contractility in vitro were significantly reduced after burn injury. These functional deficits were abolished 24 h after burn injury following local p38 MAPK inhibition. In vitro incubation of normal cardiomyocytes with homogenate from burned skin or burn serum resulted in a similar pattern of impaired cardiomyocyte contractility. These effects were reversed in normal cardiomyocytes exposed to burn skin homogenates treated topically with a p38 MAPK inhibitor. A Western blot analysis showed that cardiac p38 MAPK activation was not affected by dermal blockade of activated p38 MAPK, arguing against systemic absorption of the inhibitor and indicating the involvement of systemic cytokine signaling., Conclusion: Topical activated p38 MAPK inhibition within burned skin attenuates the release of proinflammatory mediators and prevents burn-induced cardiac dysfunction after thermal injury. These results support the inhibition of burn-wound inflammatory signaling as a new therapeutic approach to prevent potential postthermal injury multiorgan dysfunction syndrome.

The birth of molecular cardiology can be traced to the development and implementation of high-fidelity genetic approaches for manipulating the heart. Recombinant viral vector-based technology offers a highly effective approach to genetically engineer cardiac muscle in vitro and in vivo. This review highlights discoveries made in cardiac muscle physiology through the use of targeted viral-mediated genetic modification. Here the history of cardiac gene transfer technology and the strengths and limitations of viral and nonviral vectors for gene delivery are reviewed. A comprehensive account is given of the application of gene transfer technology for studying key cardiac muscle targets including Ca(2+) handling, the sarcomere, the cytoskeleton, and signaling molecules and their posttranslational modifications. The primary objective of this review is to provide a thorough analysis of gene transfer studies for understanding cardiac physiology in health and disease. By comparing results obtained from gene transfer with those obtained from transgenesis and biophysical and biochemical methodologies, this review provides a global view of cardiac structure-function with an eye towards future areas of research. The data presented here serve as a basis for discovery of new therapeutic targets for remediation of acquired and inherited cardiac diseases.

The cardiac myofilaments are composed of highly ordered arrays of proteins that coordinate cardiac contraction and relaxation in response to the rhythmic waves of [Ca(2+)] during the cardiac cycle. Several cardiac disease states are associated with altered myofilament protein interactions that contribute to cardiac dysfunction. During acute myocardial ischemia, the sensitivity of the myofilaments to activating Ca(2+) is drastically reduced, largely due to the effects of intracellular acidosis on the contractile machinery. Myofilament Ca(2+) sensitivity remains compromised in post-ischemic or "stunned" myocardium even after complete restoration of blood flow and intracellular pH, likely because of covalent modifications of or proteolytic injury to contractile proteins. In contrast, myofilament Ca(2+) sensitivity can be increased in chronic heart failure, owing in part to decreased phosphorylation of troponin I, the inhibitory subunit of the troponin regulatory complex. We highlight, in this paper, the central role of the myofilaments in the pathophysiology of each of these distinct disease entities, with a particular focus on the molecular switch protein troponin I. We also discuss the beneficial effects of a genetically engineered cardiac troponin I, with a histidine button substitution at C-terminal residue 164, for a variety of pathophysiologic conditions, including hypoxia, ischemia, ischemia-reperfusion and chronic heart failure.

Troponin I isoforms play a key role in determining myofilament Ca2+ sensitivity in cardiac muscle. The goal here was to identify domain clusters and residues that confer troponin I isoform-specific myofilament Ca2+ and pH sensitivities of contraction. Key domains/residues that contribute to troponin I isoform-specific Ca2+ and pH sensitivity were studied using gene transfer of a slow skeletal troponin I (ssTnI) template, with targeted cardiac troponin I (cTnI) residue substitutions. Substitutions in ssTnI with cognate cTnI residues R125Q, H132A, and V134E, studied both independently and together (ssTnIQAE), resulted in efficient stoichiometric replacement of endogenous myofilament cTnI in adult cardiac myocytes. In permeabilized myocytes, the pCa50 of tension ([Ca2+] required for half maximal force), and the acidosis-induced rightward shift of pCa50 were converted to the cTnI phenotype in myocytes expressing ssTnIQAE or ssTnIH132A, and there was no functionally additive effect of ssTnIQAE versus ssTnIH132A. Interestingly, only the acidosis-induced shift in Ca2+ sensitivity was comparable to cTnI in myocytes expressing ssTnIV134E, while ssTnIR125Q fully retained the ssTnI phenotype. An additional ssTnIN141H substitution, which lies within the same structural region of TnI as V134, produced a shift in myofilament Ca2+ sensitivity comparable to cTnI at physiological pH, while the acidic pH response was similar to the effect of wild-type ssTnI. Analysis of sarcomere shortening in intact adult cardiac myocytes was consistent with the force measurements. Targeted substitutions in the carboxyl portion of TnI produced residue-specific influences on myofilament Ca2+ and pH sensitivity of force and give new molecular insights into the TnI isoform dependence of myofilament function.

We previously reported that generation of the anaphylatoxin C5a is linked to the development of cardiac dysfunction in sepsis due to C5a interaction with its receptor (C5aR) on cardiomyocytes. Burn injury involves inflammatory mechanisms that can lead to C5a generation as well. In this study, we investigated the effects of C5a blockade on burn-induced cardiac dysfunction. Using a standardized rat model of full thickness scald injury, left ventricular pressures were recorded in vivo followed by in vitro assessment of sarcomere contraction of single cardiomyocytes. Left ventricular pressures in vivo and cardiomyocyte sarcomere contractility in vitro were significantly reduced following burn injury. In the presence of anti-C5a Ab, these defects were greatly attenuated 1, 6, and 12 h after burn injury and completely abolished 24 h after burn. In vitro incubation of cardiomyocytes with bacterial LPS accentuated the impaired contractility, which was partially prevented in cardiomyocytes from burned rats that had received an anti-C5a Ab. Based on Western blot analyses, real-time PCR, and immunostaining of left ventricular heart tissue, there was a significant increase in cardiomyocyte expression of C5aR after burn injury. In conclusion, an in vivo blockade of C5a attenuates burn-induced cardiac dysfunction. Further deterioration of contractility due to the exposure of cardiomyocytes to LPS was partially prevented by C5a-blockade. These results suggest a linkage between C5a and burn-induced cardiac dysfunction and a possible contribution of LPS to these events.

Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell-cell and cell-matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell-matrix, cell-cell, and intracellular cytoskeletal connections.

Vitamin D(3) deficiency enhances cardiac contraction in experimental studies, yet paradoxically this deficiency is linked to congestive heart failure in humans. Activated vitamin D(3) (1alpha,25-dihydroxyvitamin D(3)) or calcitriol, decreases peak force and activates protein kinase C (PKC) in isolated perfused hearts. However, the direct influence of this hormone on adult cardiac myocyte contractile function is not well understood. Our aim is to investigate whether 1alpha,25-dihydroxyvitamin D(3) acutely modulates contractile function via PKC activation in adult rat cardiac myocytes. Sarcomere shortening and re-lengthening were measured in electrically stimulated myocytes isolated from adult rat hearts, and the vitamin D(3) response (10(-10) to 10(-7) M) was compared to shortening observed under basal conditions. Maximum changes in sarcomere shortening and relaxation were observed with 10(-9) M 1alpha,25-dihydroxyvitamin D(3). This dose decreased peak shortening, and accelerated contraction and relaxation rates within 5 min of administration, and changes in the Ca(2+) transient contributed to the peak shortening and relaxation effects. The PKC inhibitor, bis-indolylmaleimide (500 nM) largely blocked the acute influence of the most potent dose (10(-9) M) on contractile function. While peak shortening and shortening rate returned to baseline within 30 min, there was a sustained acceleration of relaxation that continued over 60 min. Phosphorylation of the Ca(2+) regulatory proteins, phospholamban, and cardiac troponin I correlated with the accelerated relaxation observed in response to acute application of 1alpha,25-dihydroxyvitamin D(3). Accelerated relaxation continued to be observed after chronic addition of 1alpha,25-dihydroxyvitamin D(3) (e.g. 2 days), yet this sustained increase in relaxation was not associated with increased phosphorylation of phospholamban or troponin I. These results provide evidence that 1alpha,25-dihydroxyvitamin D(3) directly modulates adult myocyte contractile function, and protein kinase C plays an important signaling role in the acute response. Phosphorylation of key Ca(2+) regulatory proteins by this kinase contributes to the enhanced relaxation observed in response to acute, but not chronic calcitriol.

Obscurin is a recently identified giant multidomain muscle protein (approximately 800 kDa) whose structural and regulatory functions remain to be defined. The goal of this study was to examine the effect of obscurin gene silencing induced by RNA interference on the dynamics of myofibrillogenesis and hypertrophic response to phenylephrine in cultured rat cardiomyocytes. We found that that the adenoviral transfection of short interfering RNA (siRNA) constructs targeting the first coding exon of obscurin sequence resulted in progressive depletion of cellular obscurin. Confocal microscopy demonstrated that downregulation of obscurin expression led to the impaired assembly of new myofibrillar clusters and considerable aberrations of the normal structure of the contractile apparatus. While the establishment of the initial periodic pattern of alpha-actinin localization remained mainly unaffected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of myofibrillar bundles, leading to their abnormal bifurcation, dispersal and multiple branching. Bending of immature myofibrils, apparently associated with the loss of their rigidity, a modified titin pattern, the absence of well-formed A-bands in newly formed contractile structures as documented by a diffuse localization of sarcomeric myosin labeling, and an occasional irregular periodicity of sarcomere spacing were typical of obscurin siRNA-treated cells. These results suggest that obscurin is indispensable for spatial positioning of contractile proteins and for the structural integration and stabilization of myofibrils, especially at the stage of myosin filament incorporation and A-band assembly. This demonstrates a vital role for obscurin in myofibrillogenesis and hypertrophic growth.

A component of multiorgan dysfunction in burned patients is heart failure. Burn trauma induces cytokine synthesis of interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) which can negatively impact cardiac function. Infectious complications are common following severe burn injury. We hypothesized that burn injury and lipopolysaccharide (LPS) exposure independently influence peak cardiomyocyte contraction and cytokine secretion. Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 1, 6, 12, and 24 h after burn, cardiomyocytes were isolated and incubated with increasing LPS doses. Peak sarcomere shortening and contractile velocity parameters were recorded using a variable-rate video camera with sarcomere length detection software. Supernatants were assayed for IL-1beta, IL-6, and TNF-alpha by ELISA. Peak sarcomere shortening was decreased in the burn group at 1, 6, 12, and 24 h after burn. IL-1beta, IL-6, and TNF-alpha levels were increased in cardiomyocytes isolated 1 h after burn compared with sham controls, but returned to sham levels at 6, 12, and 24 h after burn. LPS exposure caused dose-dependent decreases in sarcomere shortening in sham and burn animals. LPS exposure did not produce increased cardiomyocyte cytokine expression. Burn injury diminished peak sarcomere shortening. Whereas exposure to LPS did not have an effect on cardiomyocyte cytokine expression, LPS significantly inhibited sarcomere shortening in a dose-dependent fashion. Combined burn and LPS exposure inhibited sarcomere shortening more than each alone. These results demonstrate that LPS exposure and burn injury independently decrease peak cardiac shortening. These decreases did not directly correlate with the levels of cytokines released in response to each stressor.

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.

Defective cardiac function during sepsis has been referred to as "cardiomyopathy of sepsis." It is known that sepsis leads to intensive activation of the complement system. In the current study, cardiac function and cardiomyocyte contractility have been evaluated in rats after cecal ligation and puncture (CLP). Significant reductions in left ventricular pressures occurred in vivo and in cardiomyocyte contractility in vitro. These defects were prevented in CLP rats given blocking antibody to C5a. Both mRNA and protein for the C5a receptor (C5aR) were constitutively expressed on cardiomyocytes; both increased as a function of time after CLP. In vitro addition of recombinant rat C5a induced dramatic contractile dysfunction in both sham and CLP cardiomyocytes, but to a consistently greater degree in cells from CLP animals. These data suggest that CLP induces C5aR on cardiomyocytes and that in vivo generation of C5a causes C5a-C5aR interaction, causing dysfunction of cardiomyocytes, resulting in compromise of cardiac performance.

Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr144 and Ser23/Ser24 clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr144 substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser23/Ser24 also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser23/Ser24 with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr144 plays an important role in the acute acceleration of relaxation, whereas Ser23/Ser24 contributes to relaxation during more prolonged activation of protein kinase C by endothelin.

Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric alpha-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for alpha-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of alpha-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of alpha-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus., (Copyright The Histochemical Society, Inc.)

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

Troponin is essential for the regulation of cardiac contraction. Troponin is a sarcomeric molecular switch, directly regulating the contractile event in concert with intracellular calcium signals. Troponin isoform switching, missense mutations, proteolytic cleavage, and posttranslational modifications are known to directly affect sarcomeric regulation. This review focuses on physiologically relevant covalent and noncovalent modifications in troponin as part of a thematic series on cardiac thin filament function in health and disease.

Our goal was to define the role of phosphorylated cardiac troponin-I in the adult myocyte contractile performance response to activated protein kinase C. In agreement with earlier work, endothelin enhanced both adult rat myocyte contractile performance and cardiac troponin-I phosphorylation. Protein kinase C participated in both responses. The role of cardiac troponin-I phosphorylation in the contractile function response to protein kinase C was further investigated using gene transfer into myocytes of troponin-I isoforms/mutants lacking one or more phosphorylation sites previously identified in purified cardiac troponin-I. Sarcomeric replacement with slow skeletal troponin-I-abrogated protein kinase C-mediated troponin-I phosphorylation. In functional studies, endothelin slowed relaxation in myocytes expressing slow skeletal troponin-I, while the relaxation rate increased in myocytes expressing cardiac troponin-I. Based on these results, acceleration of myocyte relaxation during protein kinase C activation largely depended on cardiac troponin-I phosphorylation. Experiments with troponin-I isoform chimeras provided evidence that phosphorylation sites in the amino portion of cardiac troponin I-mediated the protein kinase C acceleration of relaxation. The cardiac troponin-I Thr-144 phosphorylation site identified in earlier biochemical studies was not significantly phosphorylated during the acute contractile response. Thus, amino-terminal protein kinase C-dependent phosphorylation sites in cardiac troponin-I are likely responsible for the accelerated relaxation observed in adult myocytes.

Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40-50% of native Tm or TnT were specifically replaced. The Ca(2+) sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium.

Thin filament proteins play an essential role in the regulation of myocardial pressure development. Within the thin filament of the sarcomere, troponin I (TnI) plays a key role in regulating the Ca(2+) sensitivity of force. During myocardial development, there is a transition in TnI isoform expression from the slow skeletal isoform (ssTnI) in embryonic/fetal myocardium to the cardiac isoform (cTnI) expressed in adult hearts. Over a similar developmental time window, the calcium sensitivity of force development also decreases. Gene transfer of ssTnI, and chimeras derived from ssTnI and cTnI, into adult ventricular myocytes have provided insights into the isoform-specific domains of TnI responsible for differentially influencing myofilament Ca(2+) sensitivity. Two separate isoform-specific regions, located in the carboxyl- and amino-portions of the protein, have been identified by comparing Ca(2+)-activated isometric tension in myocytes expressing the TnI isoforms or chimeras. The carboxyl-portion of TnI also contributes to isoform-dependent differences in myofilament sensitivity to acidic pH, which ensues during several myocardial disease states. In contrast, the diminished Ca(2+) sensitivity observed in response to beta-adrenergic-mediated phosphorylation of cardiac TnI requires the amino-portion of the cardiac TnI isoform yet, does not depend on the presence of a specific isoform in the carboxyl-region of TnI. Recent studies with a mutation linked to hypertrophic cardiomyopathy have demonstrated that changes in protein charge also influence the ability of TnI isoforms to regulate myofilament Ca(2+) sensitivity. Information gained from these, and future studies on more localized and specific changes in the amino acid sequence, may one day lead to the use of genetically engineered TnI for therapeutic manipulation of contractile function.

The heightened Ca2+ sensitivity of force found with hypertrophic cardiomyopathy (HCM)-associated mutant cardiac troponin I (cTnIR145G; R146G in rodents) has been postulated to be an underlying cause of hypertrophic growth and premature sudden death in humans and in animal models of the disease. Expression of slow skeletal TnI (ssTnI), a TnI isoform naturally expressed in developing heart, also increases myofilament Ca2+ sensitivity, yet its expression in transgenic mouse hearts is not associated with overt cardiac disease. Gene transfer of TnI isoforms or mutants into adult cardiac myocytes is used here to ascertain if expression levels or functional differences between HCM TnI and ssTnI could help explain these divergent organ-level effects. Results showed significantly reduced myofilament incorporation of cTnIR146G compared with ssTnI or wild-type cTnI. Despite differences in myofilament incorporation, ssTnI and cTnIR146G expression each resulted in enhanced myofilament tension in response to submaximal Ca2+ under physiological ionic conditions. Myofilament expression of an analogous HCM mutation in ssTnI (ssTnIR115G) did not further increase myofilament Ca2+ sensitivity of tension compared with ssTnI. In contrast, there was a divergent response under acidic pH conditions, a condition associated with the myocardial ischemia that often accompanies hypertrophic cardiomyopathy. The acidic pH-induced decrease in myofilament Ca2+ sensitivity was significantly greater in myocytes expressing cTnIR146G and ssTnIR115G compared with ssTnI. These results suggest that differences in pH sensitivities between wild-type ssTnI and mutant TnI proteins may be one factor in helping explain the divergent organ and organismal outcomes in TnI HCM- and ssTnI-expressing mice.

Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in proteins of the cardiac contractile apparatus, including alpha-tropomyosin (Tm). Mice expressing alphaTm in the heart were developed to determine the effects of FHC mutant Tm on cardiac structure and function from single cardiac myocytes to whole organ function in vivo. Expression of E180G mutant Tm did not produce cardiac hypertrophy or detectable changes in cardiac muscle morphology. However, E180G mutant Tm expression increased the Ca2+ sensitivity of force production in single cardiac myocytes in a transgene expression-dependent manner. Contractile dysfunction in single myocytes manifested organ level dysfunction, as conductance-micromanometry showed E180G Tm mice had significantly slowed relaxation (diastolic dysfunction) under physiological conditions. The diastolic dysfunction in E180G Tm mice was no longer evident during beta-blockade because propranolol eliminated the effect of E180G Tm to slow myocardial relaxation. Cellular and organ level dysfunction were evident in E180G Tm mice in the absence of significant cardiac structural abnormalities normally associated with FHC. These findings therefore suggest that diastolic dysfunction in FHC may be a direct consequence of FHC mutant protein expression. In addition, because diastolic dysfunction in E180G Tm mice is dependent on inotropic status, cardiovascular stress may play an important role in FHC pathogenesis.