Intact Papillary Muscles Research Articles

Abstract 11144: Mitral Valve Leaflet Strains Are Unphysiologically Altered After Mitraclip to Repair Functional Mitral Regurgitation

Introduction: Transcatheter edge-to-edge repair (TEER) by approximating the leaflet edges with the MitraClip system ( Fig. A ) is rapidly gaining momentum as the primary therapy for repair of functional mitral regurgitation. Though effective acutely, the unphysiological mechanics that TEER imposes on the valve can be substantial and effect durability of the repair. In this study, we used stereo digital image correlation (sDIC) to quantify the leaflet strains that TEER imposes on the mitral valve in the repair of FMR. Hypothesis: TEER imposes an unphysiological diastolic and early systolic strain on the mitral valve leaflets, which may be associated with poor long-term outcomes. Methods: MVs with intact papillary muscles (PMs) were isolated from porcine hearts and mounted within an acrylic left ventricle chamber. A speckle pattern was imprinted on the valve leaflet surface and a sDIC system with two high-speed cameras imaging at 250 fps recorded synchronized image sequences upon pressurization of the chamber to 120 mm Hg in three different configurations: baseline/healthy, FMR/tethered valve configuration, and with TEER/MitraClip. Results: FMR configuration ( Fig. A2 ) did not alter the peak systolic leaflet strains but elevated the diastolic leaflet strains. The time to peak strain was delayed as well in the FMR configuration. TEER reduced the overall systolic strains on the leaflet from the disease state, but the diastolic strains were further elevated compared to FMR condition. Sub-regional analysis depicted that the leaflet edge section where the TEER was applied was under constantly high strain throughout the cardiac cycle, without an unloading phase. Conclusions: TEER imposes a supraphysiological strain field on the mitral valve leaflet when used to repair FMR. The impact of such strains is not yet known, but may be associated with leaflet remodeling and the 2% of the procedures in which single leaflet detachment has been reported.

Cardiac‐specific Deletion of Filamin C Decouples Myocyte Contractility and Calcium Handling in Adult Mice

Costameres are sub‐membranous protein complexes in striated muscle that play roles in mechanical signal transduction and force transmission between the Z‐disc of the sarcomere and the extracellular matrix. In cardiac myocytes (CMs), the cytoskeletal protein Filamin C (FLNC) interacts with multiple proteins in the Z‐disc and the costamere, suggesting that it contributes to costamere‐based mechanotransduction in the heart. Moreover, mutations in the gene that encodes FLNC have been associated with multiple forms of human cardiomyopathies. To investigate the role of FLNC in cardiac mechanosignaling and pathological remodeling, we employed a recently developed murine model of inducible and cardiac‐specific FLNC knockout (FLNC‐icKO). Previous studies with this mouse model used echocardiographic measurements of adult FLNC‐icKO mice to reveal a severe dilated cardiomyopathy (DCM) phenotype by 2 weeks post‐induction, including significantly reduced ventricular fractional shortening. Correspondingly, we found that intact papillary muscles dissected from adult FLNC‐icKO hearts have significantly reduced twitch force compared to those dissected from wild‐type (WT) hearts. Interestingly, while the fractional shortening of isolated CMs from FLNC‐icKO hearts is also significantly reduced compared to WT CMs, the calcium transient (peak amplitude and duration) during a twitch is not different between groups. These results suggest that contractility and calcium handling in FLNC‐null hearts are decoupled. Potential mechanisms that may be responsible for this apparent decoupling are a disruption in force transmission between the sarcomere and the cell membrane, an increased myofilament lattice spacing, or altered calcium sensitivity of the myofilaments upon the deletion of FLNC in CMs.Support or Funding InformationFunding: HL007444‐36 to JDP, HL144872 to JC, and HL137100 to ADM.

Cardiac Myosin Binding Protein-C Phosphorylation Mitigates Age-Related Cardiac Dysfunction: Hope for Better Aging?

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation prevents aging-related cardiac dysfunction. We tested this hypothesis by aging genetic mouse models of hypophosphorylated cMyBP-C, wild-type equivalent, and phosphorylated-mimetic cMyBP-C for 18 to 20months. Phosphorylated-mimetic cMyBP-C mice exhibited better survival, better preservation of systolic and diastolic functions, and unchanging wall thickness. Wild-type equivalent mice showed decreasing cMyBP-C phosphorylation along with worsening cardiac function and hypertrophy approaching those found in hypophosphorylated cMyBP-C mice. Intact papillary muscle experiments suggested that cMyBP-C phosphorylation increased cross-bridge detachment rates as the underlying mechanism. Thus, phosphorylating cMyBP-C is a novel mechanism with potential to treat aging-related cardiacdysfunction.

Abstract 621: Cardiac Myosin Binding Protein-C Phosphorylation in Ser-273 and Ser-282 is Critical to Maintain Cardiac Function During Aging

Background/hypothesis: Heart failure (HF) afflicts 6.2M Americans as of 2019 and its prevalence is expected to increase due to a growing aging population. HF progression involves depression in contractility and relaxation, which are strongly controlled by phosphorylation of cardiac myosin binding protein-C (cMyBP-C). We hypothesize that phosphorylated cMyBP-C supports normal heart function during aging. Methods: We aged mouse models of cMyBP-C de-phosphorylation mimetic (t3SA: S273A/S282A/S302A mutations), phosphorylation mimetic (t3SD: S273D/S282D/S302D mutations) and WT equivalent expressed onto cMyBP-C(-/-) background (tWT). Results: Western blotting (N=16, 2-24 months) showed that cMyBP-C phosphorylation decreases with aging at S273 and S282, showing strong Pearson correlations with age (see figure; S273P r= -0.931, p<0.0001; S282P r=-0.868, p<0.00001). Simultaneous force and [Ca 2+ ] i measurements on intact papillary muscles showed that phosphorylation-mimetic t3SD has greatest dFR=(-dF/dt)min/(+dF/dt)max, signifying better lusitropy with similar calcium decay rate constant (k Ca ) as others. Echocardiography at 3, 12, 15, 18 months showed that t3SD has preserved ejection fraction, faster peak myocardial relaxation velocity e’ and preserved posterior ventricular wall thickness with aging. Moreover, t3SD demonstrated superior 600-days survival than others (t3SD 87.5% n=96; tWT 69.3% n=122; t3SA 53.2% n=97; overall LogRank p=0.0002244). Conclusions: De-phosphorylation of cMyBP-C at S273 and S282 contributes to worsening of cardiac function with aging. Conversely, phosphorylated cMyBP-C mitigates aging-related cardiac dysfunction.

NNOS splice variants differentially regulate myofilament function but are dispensable for intracellular calcium and force transients in cardiac papillary muscles.

Cardiac muscle expresses three neuronal nitric oxide synthase (nNOS) splice variants: nNOSα, nNOSμ and nNOSβ. The functions of these nNOS splice variants in cardiac muscle, particularly myofilament-associated nNOSβ are unclear. To decipher cardiac nNOS splice variant function we investigated myofilament function and intracellular calcium and force transients in demembranated and intact papillary muscles from two lines of nNOS knockout mice. The first line (KN1) lacks nNOSα and nNOSμ. The second line (KN2) lacks active nNOSα, nNOSμ and nNOSβ. Demembranated KN1 papillary muscles exhibited reduced myofilament ATPase activity (-35%) and specific force (-10%) relative to controls. Demembranated KN2 muscles exhibited a smaller decrease in myofilament ATPase activity (-21%), but a greater reduction in specific force (-26%) relative to controls. Myofilament calcium sensitivity in demembranated KN1 and KN2 papillary muscles was similar to controls. Thus, papillary muscle-expressed nNOS splice variants are necessary for control levels of myofilament ATPase activity and force generation, but dispensable for myofilament calcium sensitivity. The greater reduction in myofilament ATPase relative to specific force in KN1, but not KN2 muscle, reduced the energy cost of muscle contraction, suggesting that nNOSβ increased the energetic efficiency of contraction in the absence of nNOSμ and nNOSα. Analyses of intact KN1 and KN2 papillary muscles showed that both intracellular calcium transients and their evoked force transients were similar to controls at stimulation frequencies between 1 and 3 Hz. Therefore, nNOS was dispensable for baseline excitation-contraction coupling. In summary, these data suggest that nNOS splice variants differentially regulate myofilament function, but not baseline calcium handling in papillary muscles. More importantly, they suggest that nNOSβ is a novel modulator of myofilament function, and ultimately the energetic efficiency of cardiac papillary muscle contraction.

Abstract 17263: Cardiac Myosin Binding Protein-C Phosphorylation Mitigates Pressure-Overload Heart Failure Despite Alterations in Calcium Handling

Heart failure with reduced ejection (HFrEF) fraction exhibits both systolic and diastolic dysfunctions; therefore, treatments that improve contractility and lusitropy will be helpful. Cardiac myosin binding protein-C (cMyBP-C), which is a thick filament protein, can increase rate of cross-bridge cycling by its phosphorylation status to enhance contractility and lusitropy. Consequently, we hypothesized that cMyBP-C phosphorylation mitigates HFrEF by preserving systolic and diastolic functions. We tested our idea by challenging WT cMyBP-C(tWT), phosphorylation-deficient cMyBP-C(t3SA):S273A/S282A/S302A, and phosphorylation-mimetic cMyBP-C(t3SD):S273D/S282D/S302D mouse models with trans-aortic constriction (TAC) to induce pressure-overload HFrEF. Survival at 5 weeks showed notable differences (tWT 75.6±6.7% n=41, t3SA 53.4±6.7% n=68, t3SD 92.1±4.4% n=38, p=0.00037). Serial echocardiography showed that cardiac structure and function deteriorated for all mouse models. In comparison to tWT at post-TAC week-5, t3SA demonstrated ventricular dilation and reduced ejection fraction (EF); conversely, t3SD exhibited better preservation of diastolic function (smaller E/Ea, where E=blood flow Doppler and Ea=tissue Doppler of peak myocardial relaxation velocity). Electrically paced intact papillary muscles from t3SA and t3SD demonstrated slower intracellular calcium decay (smaller decay rate constant kCa). Western blotting showed: (1) decreased calcium-ATPase (SERCA2a) expression in t3SA and t3SD, (2) lowest ryanodine receptor (RyR2) expression in t3SD, and (3) decreased calcium calmodulin kinase-2 phosphorylation (pCaMK2) only in t3SD. These calcium handling protein alterations likely prevented stronger rescue by cMyBP-C phosphorylation mimetic. Thus, cMyBP-C phosphorylation mimetic mitigated TAC-induced HFrEF (better survival and preservation of diastolic function) despite depression of calcium handling proteins.

Abstract 231: Cardiac Myosin Binding Protein C Couples Cross-Bridge Cycling to Calcium Transients to Provide Normal Cardiac Function

A 40-year old American has a 20% chance of developing heart failure (HF) in his/her lifetime. Although cross-bridge cycling forms the basis for contraction and relaxation of the heart, mechanisms that modulate cross-bridge cycling are not completely understood, nor are the alterations in these processes in HF. Cardiac myosin binding protein-C (MyBPC3) is a component of heart muscle that is believed to regulate cross-bridge cycling kinetics. We hypothesize that MyBPC3 tunes cross-bridge cycling to the [Ca2+]i transient to produce normal cardiac function, which we tested using our MyBPC3 knockout (KO) mouse. Echocardiography showed that MyBPC3(KO) hearts exhibited abbreviated aortic ejection durations (AED) and AED/(period between heart beats) ratio compared to WT. MyBPC3(KO) hearts also had smaller velocity-time integrals which indicate smaller stroke volumes as a result of abbreviated ejection. Brain natriuretic peptide expression level, heart weight/body weight, and lung weight/body weight were all increased in MyBPC3(KO) mice, consistent with HF. MyBPC3(KO) mice also exhibit increased 1-year mortality. We used simultaneous twitch force and intracellular calcium [Ca2+]i measurements on intact papillary muscles to evaluate the consequences of losing cross-bridge cycling regulation on force response to changing [Ca2+]i transients. MyBPC3 (KO) myocardium exhibited abbreviated contractions despite slower calcium transients and an inability to accelerate relaxation with respect to peak contraction. Therefore, loss of MyBPC3-mediated coupling of cross-bridge cycling to the [Ca2+]i transient was associated with contractile dysfunction and HF.

Reduced Efficiency of Intact Papillary Muscles in ACTC E99K Transgenic Mouse Heart

The E99K transgenic (TG) mouse expresses mutant cardiac actin as reported in human patients being diagnosed with hypertrophic cardiomyopathy (HCM) predominantly in the apex. The mouse has recapitulated many phenotypes as in human patients including sudden cardiac death, apical hypertrophy, fibrosis, myocyte disarray and higher Ca2+-sensitivity. We are now reporting a reduced efficiency of the mutant cardiac muscle comparing with their non-transgenic (NTG) litter mates. Intact papillary muscle used in the experiments and the efficiency during contractions was measured with a protocol of 40 twitches in 20s (27oC). The work and the heat production were measured during each set of 40 twitches and during runs with no stimulation as control. To mimic the cardiac cycle, each stimulus was followed by an isometric period (isovolumic contraction) of 0.12s, shortening by 10%L0, an isometric period (isovolumic relaxation), and lengthening by 10% L0. Four movement velocities, 0.5, 0.67, 1 and 2 L0/s were tested. The isovoluminc relaxation periods were adjusted accordingly. Net work was calculated as the integral of active force and length change. Heat production, an index of metabolic cost, was calculated from temperature change measured with a thermopile of constantan-chromel thermocouples. Muscle of TG mouse (n=12) did more work/mg muscle (33 ± 1, mJ/g) than muscles from NTG littermates (19 ± 4, mJ/g, n=11), with disproportionately more total energy, work + heat (263 ± 10, mJ/g) than NTG (114 ± 18, mJ/g). Thus E99K muscle was less efficient (= work/total energy), 0.127 ± 0.005 than NTG muscle, 0.166 ± 0.015. Considering the higher Ca2+-sensitivity detected in myofibril and skinned papillary muscle levels, more energy was likely used for active transport of ions such as Ca2+.

Human Heart Slices - a Novel Multicellular System Suitable for Electrophysiological and Pharmacological Studies

Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human cardiac tissue. Here, we describe and characterise a novel, simple and reproducible preparation suitable for studying human cardiac tissue at the multicellular level, the living human cardiac slice.Vibratome-cut slices (350 μm thick) were prepared from left ventricular transmural biopsies (5x5 mm) obtained from end-stage heart failure patients undergoing transplant or left ventricular assist device implantation. One biopsy was sufficient to generate >15 slices. Histology revealed that slices cut in parallel to the epicardial surface had predominantly longitudinal muscle fibre orientation. ATP/ADP and phosphocreatine/creatine ratios (3.32±0.61 & 1.37±0.31; n=5 slices/3 hearts) measured by HPLC were comparable to intact organ values.Electrical activity and its propagation in response to electrical stimulation (1Hz) were recorded using a multi-electrode array system. Field potential duration (FPD) remained stable and could be recorded for up to 8h.Mean FPD was 467±4 ms (n=42/10), similar to previously reported data using ventricular wedges.Longitudinal conduction velocity (CV) was faster than transversal CV (51±3 & 20±1 cm/sec, n=19/7, p<0.0001), with an anisotropic ratio of 2.5:1.Ikr blocker E4031 (1 μM) and Iks blocker Chromanol 293B (20 μM) prolonged FPD by 64±3% (n=20/7, p<0.001) and 22±2% (n=12/5, p<0.001) respectively, similar to previous results using intact papillary muscles. The non-specific K+ channel blocker 4-aminopyridine (1mM) prolonged FPD by 47±2% (n=6/2, p<0.001).Our results show that cardiac slices from human biopsies are a reproducible preparation that remains stable for several hours in vitro and has structural, electrophysiological, pharmacological and metabolic properties of native myocardium. Cardiac slices offer a novel multicellular system suitable for the study of heart failure and therapy.

The FHC Mutation D166V in the Cardiac Muscle Regulatory Light-Chain Causes Both a Diastolic and Systolic Dysfunction in Mouse Cardiac Muscle Performance

Previously we showed that the D166V (aspartic acid to valine) mutation in the myosin regulatory light-chain (RLC), implicated in familial hypertrophic cardiomyopathy, was associated with a decrease in the rate that force-generating cross-bridges dissociate (g) and an increased Ca2+ sensitivity of Ca2+ activated skinned fiber force and ATPase (Kerrick et al. FASEB J. 2009, 23:855-65). Here, we tested the hypothesis that the previous findings most likely manifested a diastolic dysfunction of the mutated myocardium. Transgenic intact papillary muscle fibers from D166V and WT mice were stimulated to contract at different frequencies, and force and intracellular Ca2+ were measured simultaneously. Results showed that as frequency of stimulation increased, the end-twitch force increased greatly in transgenic D166V papillary muscles compared to WT, demonstrating diastolic dysfunction. The end-twitch intracellular Ca2+ increased similarly with increasing frequency in both D166V and WT mice, as expected. However, it was found that the twitch force in D166V muscles initially increased and then decreased with increasing frequency of stimulation as did the amplitude of the intracellular Ca2+ transient, a possible explanation for why force amplitude decreased at higher frequencies. Another explanation for a decrease in force at high contraction frequencies is that RLC phosphorylation, activated by the increasing levels of diastolic Ca2+, may affect force differently in D166V (lowers force) than in WT muscles. In contrast to D166V, WT papillary muscle twitch force increased linearly over the same range of frequencies studied reaching a maximum force far exceeding that of D166V muscles. In summary the FHC D166V-RLC mutation of cardiac muscle would be expected to cause both a diastolic and systolic dysfunction in cardiac muscle performance. Supported by AHA 10GRNT4450042(WGK) and 10POST3420009(PM), and NIH-HL090786(DSC).

Functional effects of a tropomyosin mutation linked to FHC contribute to maladaptation during acidosis

Familial hypertrophic cardiomyopathy (FHC) is a leading cause of sudden cardiac death among young athletes but the functional effects of the myofilament mutations during FHC-associated ischemia and acidosis, due in part to increased extravascular compressive forces and microvascular dysfunction, are not well characterized. We tested the hypothesis that the FHC-linked tropomyosin (Tm) mutation Tm-E180G alters the contractile response to acidosis via increased myofilament Ca2+ sensitivity. Intact papillary muscles from transgenic (TG) mice expressing Tm-E180G and exposed to acidic conditions (pH 6.9) exhibited a significantly smaller decrease in normalized isometric tension compared to non-transgenic (NTG) preparations. Times to peak tension and to 90% of twitch force relaxation in TG papillary muscles were significantly prolonged. Intact single ventricular TG myocytes demonstrated significantly less inhibition of unloaded shortening during moderate acidosis (pH 7.1) than NTG myocytes. The peak Ca2+ transients were not different for TG or NTG at any pH tested. The time constant of re-lengthening was slower in TG myocytes, but not the rate of Ca2+ decline. TG detergent-extracted fibers demonstrated increased Ca2+ sensitivity of force and maximal tension compared to NTG at both normal and acidic pH (pH 6.5). Tm phosphorylation was not different between TG and NTG muscles at either pH. Our data indicate that acidic pH diminished developed force in hearts of TG mice less than in NTG due to their inherently increased myofilament Ca2+ sensitivity, thus potentially contributing to altered energy demands and increased propensity for contractile dysfunction.

Mechanical properties of passive myocardium: experiment and mathematical model

We present here a 3D mathematical model of rheological properties of a morphofunctional unit of myocardium as example of biological tissue. The model consists of longitudinal and transverse elastic elements and inclined viscoelastic elements connected pivotally without friction. The parameters of viscosity and elasticity of the structural elements of the model do not depend on the magnitude of deformation. The model adequately describes nonlinear viscoelastic behavior of isolated samples of passive myocardium both in static condition and under dynamic loading. The simulation data fit the experiment very well both for intact rat papillary muscle and for a decellularized specimen.

Traumatic Tricuspid Insufficiency with Intact Chordae and Papillary Muscles: A Case Report

Traumatic Tricuspid Insufficiency with Intact Chordae and Papillary Muscles: A Case Report

Is Slower Myosin Cross-Bridge Kinetics in Tg-D166V Preparations Due to Decreased Myosin RLC Phosphorylation?

In this report we have investigated a particularly malignant phenotype of Familial Hypertrophic Cardiomyopathy (FHC) associated with the 166 Aspartic Acid to Valine (D166V) mutation in the ventricular myosin regulatory light chain (RLC). We show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from transgenic (Tg)-D166V compared to Tg-WT mice. A single molecule approach was taken where the fluorescence anisotropy of rhodamine phalloidin labeled actin protomers was measured in cardiac myofibrils undergoing isometric contraction. Orientation of an actin molecule oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rates of cross-bridge attachment as well as cross-bridge dissociation were significantly decreased in isometrically contracting Tg-D166V myofibrils (binding, 1.4 s−1; detachment, 1.2 s−1) compared to Tg-WT myofibrils (binding, 3 s−1; detachment, 1.3 s−1). The duty ratio of the cross-bridge cycle, equal to the fraction of the total cycle time that cross-bridge remains attached to actin, was 47% inTg-D166V myofibrils and 30% in Tg-WT. Immunoblotting of cardiac myofibrils used for kinetics studies demonstrated a large reduction in RLC phosphorylation in Tg-D166V vs. Tg-WT myofibrils. These data are in accord with our previous findings in skinned and intact papillary muscles showing slower fiber kinetics and prolonged force transients in Tg-D166V fibers compared to Tg-WT preparations (Kerrick et al., FASEB J. 23: 855, 2009). Similarly, the level of RLC phosphorylation in muscle extracts from Tg-D166V ventricles was decreased. Our cellular and single molecule data suggest that a mutation-dependent decrease in RLC phosphorylationcould initiate the slower kinetics of the D166V cross-bridgesand ultimately lead to abnormal cardiac muscle contraction. Supported by NIH-HL071778 (DSC), NIH-AR048622 (JB) and NIH-HL090786 (to JB and DSC).

Left Ventricular and Myocardial Function in Mice Expressing Constitutively Pseudophosphorylated Cardiac Troponin I

Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction. Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnI(PKC-P)) expressing mutant cTnI (S43E, S45E, T144E). Two-dimensional gel analysis showed 7.2+/-0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model-based analysis for integrative interpretation. Compared to wild-type mice, cTnI(PKC-P) mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, F(max)). Additionally, cTnI(PKC-P) mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnI(PKC-P) mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnI(PKC-P) mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state. A modest increase in PKC-induced cTnI phosphorylation ( approximately 7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).

Surgical Management of Ischemic Mitral Regurgitation

Case presentation: A 62-year-old man visited his cardiologist describing a 6-month history of increasing fatigue and shortness of breath on exertion. His medical history included an inferoposterior myocardial infarction (MI) 3 years previously that was treated with drug-eluting stents to his left circumflex and right coronary arteries. The result of physical examination was remarkable for crackles audible throughout the lower half of both lung fields, an elevated jugular venous pulse to the angle of the jaw, and a grade 3/6 holosystolic murmur loudest at the apex with radiation to the left axilla. An echocardiogram revealed a left ventricular ejection fraction (LVEF) of 15% to 20%, severe mitral regurgitation (MR), and a left ventricular end systolic dimension of 70 mm. The patient underwent a repeat cardiac angiogram that revealed patent stents in the circumflex and right coronary arteries and no other significant stenoses. The decision was made to surgically correct the patient’s MR with a mitral valve operation. He was brought to the operating room, where he was given general anesthesia, and a transesophageal echocardiogram was performed. Examination of the mitral valve revealed poor leaflet coaptation with a degree of leaflet tethering that resulted in severe MR. Inasmuch as the valve leaflets appeared structurally normal, a mitral valve repair procedure using a complete rigid annuloplasty ring was performed. A postoperative echocardiogram revealed trace MR, and the patient experienced no postoperative complications. This patient is a typical example of a growing problem in our aging population, namely, ischemic mitral regurgitation (IMR). IMR clearly has a negative impact on survival in patients with coronary artery disease, even in patients with mild to moderate MR; greater degrees of MR portend an even worse prognosis.1 With almost a fifth of patients who experience an MI followed by the development of IMR,2,3 and over …

FHC-linked Mutations in the Myosin Regulatory Light Chain Interfere with RLC Phosphorylation in Transgenic Mice

Previous studies have shown that MLCK-phosphorylation of the ventricular regulatory light chain (RLC) increases myofilament Ca2+ sensitivity, maximal level of force and rate of tension development thereby enhancing the systolic function of the heart. We have investigated the effect of FHC (familial hypertrophic cardiomyopathy) mutations in the myosin RLC on its phosphorylation measured in rapidly frozen ventricular samples from transgenic (Tg) mice. We observe that the Aspartate to Valine substitution in Tg-D166V mice and Arginine to Glutamine in Tg-R58Q mice result in a decreased phosphorylation of RLC detected in left ventricular samples from Tg-mutant compared to Tg-WT mice expressing the human ventricular RLC. The level of RLC phosphorylation was determined by Western blotting utilizing human specific phospho-RLC antibodies (gift from Dr. N. Epstein, NIH). Our data from skinned Tg-D166V and Tg-R58Q papillary muscle fibers show that both RLC mutations lead to a decreased maximal level of force and to slower kinetics of force generating myosin cross-bridges compared to WT fibers. Studies in intact papillary muscle fibers show prolonged force transients for both D166V and R58Q mutants. In addition, the hearts of aging mutant mice demonstrate histopathological changes and frequent occurrences of fibrotic lesions. Clinical studies revealed that both D166V and R58Q mutations are associated with severe FHC phenotypes with multiple cases of sudden cardiac death. Our results suggest that phosphorylation of RLC plays an important role in regulating cardiac function and its deficit may contribute to malignant FHC phenotypes. Reduced RLC phosphorylation observed in the D166V and R58Q hearts correlates with our cellular findings and could be responsible for delayed force transients, slower cross-bridge kinetics and decreased force observed in Tg-D166V and Tg-R58Q papillary muscle fibers. Supported by NIH- HL071778 (D.S-C.).

Diastolic dysfunction in familial hypertrophic cardiomyopathy transgenic model mice

Several mutations in the ventricular myosin regulatory light chain (RLC) were identified to cause familial hypertrophic cardiomyopathy (FHC). Based on our previous cellular findings showing delayed calcium transients in electrically stimulated intact papillary muscle fibres from transgenic Tg-R58Q and Tg-N47K mice and, in addition, prolonged force transients in Tg-R58Q fibres, we hypothesized that the malignant FHC phenotype associated with the R58Q mutation is most likely related to diastolic dysfunction. Cardiac morphology and in vivo haemodynamics by echocardiography as well as cardiac function in isolated perfused working hearts were assessed in transgenic (Tg) mutant mice. The ATPase-pCa relationship was determined in myofibrils isolated from Tg mouse hearts. In addition, the effect of both mutations on RLC phosphorylation was examined in rapidly frozen ventricular samples from Tg mice. Significantly, decreased cardiac function was observed in isolated perfused working hearts from both Tg-R58Q and Tg-N47K mice. However, echocardiographic examination showed significant alterations in diastolic transmitral velocities and deceleration time only in Tg-R58Q myocardium. Likewise, changes in Ca(2+) sensitivity, cooperativity, and an elevated level of ATPase activity at low [Ca(2+)] were only observed in myofibrils from Tg-R58Q mice. In addition, the R58Q mutation and not the N47K led to reduced RLC phosphorylation in Tg ventricles. Our results suggest that the N47K and R58Q mutations may act through similar mechanisms, leading to compensatory hypertrophy of the functionally compromised myocardium, but the malignant R58Q phenotype is most likely associated with more severe alterations in cardiac performance manifested as impaired relaxation and global diastolic dysfunction. At the molecular level, we suggest that by reducing the phosphorylation of RLC, the R58Q mutation decreases the kinetics of myosin cross-bridges, leading to an increased myofilament calcium sensitivity and to overall changes in intracellular Ca(2+) homeostasis.

Abstract 5403: Protein Kinase C-Induced Troponin I Phosphorylation Causes Changes in Cardiac Contractile Function but not the Intracellular Calcium Transient

Both intracellular calcium transients ([Ca] i ) and myofilament properties determine cardiac muscle contractile force. Transgenic mouse models created to perturb specific myofilament proteins often cause a compensatory change in [Ca] i , which confounds the assessment of myofilament structure-function relationships. We have created a new transgenic mouse that has all three protein kinase C (PKC) phosphorylation sites on cardiac troponin I (cTnI) mutated to glutamic acid, rendering these sites constitutively pseudo-phosphorylated. Our goal was to determine the effects of this mutation on cardiac muscle contractile function and whether these effects would be concurrent with changes in the [Ca] i . Two sets of studies were conducted: skinned muscle fiber experiments to characterize the steady-state force-pCa relationships at sarcomere lengths of 1.9 and 2.3 μm and right ventricular papillary muscle experiments to characterize the peak developed force (F dev )-muscle length (L) relationships and [Ca] i (fura-5F calcium dye, emission: 510 nm, excitation: 340 and 380 nm, R = [emission fluorescence 340 ]/[emission fluorescence 380 ]). In skinned fibers, there was a significant decrease in maximally activated force (i.e., force at pCa 4.33) in transgenic mice (Wild-Type, WT (n = 7): 64.4± 8.0, Transgenic, TG (n = 6): 42.6±6.8 mN•mm −2 , P = 0.004), without any changes in calcium sensitivity or cooperativity (Hill coefficient). In intact papillary muscles, TG mice showed a decrease in F dev and slowed relaxation for all muscle lengths examined (F dev @ 100% L max , WT (n = 5): 9.3±3.5, TG (n = 6): 4.2±1.6 mN•mm −2 , P = 0.005; dF/dt min @ 100% L max , WT: −136±32, TG: −74±38 mN•mm −2 •s −1 , P = 0.002). In contrast, [Ca] i was unaltered in TG mice at all muscle lengths examined ([Ca] i amplitude as quantified by R systole / R diaastole , WT: 1.62±0.07, TG: 1.48±0.22; [Ca] i relaxation rate d R /dt min , WT: −96±37, TG: −64±30 s −1 ). Thus, PKC-induced TnI phosphorylation affects cardiac muscle contraction (reduced force magnitude and slowed relaxation) via changes in the myofilament properties (activation and/or crossbridge dynamics), and these contractile effects are not related to any changes in the intracellular calcium transient.

Abstract 306: Engineered Neonatal Rat Cardiac Tissue Models Immature in vivo Myocardium

Correlating contractile function, cellular, and molecular events in the immature heart is hampered by the limited availability of easily studied models. 2D cultures provide molecular data but can not replicate in vivo mechanical events. Isolated perfused hearts provide contractile data, but gene expression manipulation and molecular studies are limited. Here we demonstrate the utility of a 3D neonatal cardiomyocyte culture to model immature native myocardium. Isolated cardiomyocytes from day-old Sprague-Dawley rat pups were mixed with collagen and matrigel, and cast into cylindrical constructs (termed NECT). After seven days in culture NECT force-strain, force-frequency, and calcium transient data were collected. Identical data were collected from aged matched intact papillary muscle. RNA and protein were isolated from NECT and papillary muscle to assess sarcomeric proteins and quantitate developmentally regulated gene expression. Transcription of β-actin and cardiac α-actin were lower in NECT vs. native ( P &lt;0.001; N=8). Myosin heavy chain α and β isoform ratios and content were similar between NECT and native while metabolic genes GLUT1 and MCAD were increased, and GLUT4 and PDK4 decreased ( P &lt; .05) . Major sarcomeric proteins were well represented in NECT while regulatory proteins MyBPC and TnI were decreased. Peak developed force and calcium transients were similar between NECT and native, while the rate of force development and relaxation were accelerated in the NECT (Table ). 3D culture of neonatal cardiomyocytes, NECT, replicates important structural and functional features of immature native myocardium. The in vitro investigation of 3D NECT represents a unique opportunity to study electromechanical, metabolic, and endocrine factors that influence myocardial maturation. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania &amp; West Virginia). Comparison of Force and Calcium Transients in NECT