EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis. This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 °C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 µm diameter illumination spot, corresponding to the area of the contractility measurements.

Large-Scale Contractility Measurements Reveal Large Atrioventricular and Subtle Interventricular Differences in Cultured Unloaded Rat Cardiomyocytes.

The chambers of the heart fulfill different hemodynamic functions, which are reflected in their structural and contractile properties. While the atria are highly elastic to allow filling from the venous system, the ventricles need to be able to produce sufficiently high pressures to eject blood into the circulation. The right ventricle (RV) pumps into the low pressure pulmonary circulation, while the left ventricle (LV) needs to overcome the high pressure of the systemic circulation. It is incompletely understood whether these differences can be explained by the contractile differences at the level of the individual cardiomyocytes of the chambers. We addressed this by isolating cardiomyocytes from atria, RV, LV, and interventricular septum (IVS) of five healthy wild-type rats. Using a high-throughput contractility set-up, we measured contractile function of 2,043 cells after overnight culture. Compared to ventricular cardiomyocytes, atrial cells showed a twofold lower contraction amplitude and 1.4- to 1.7-fold slower kinetics of contraction and relaxation. The interventricular differences in contractile function were much smaller; RV cells displayed 12-13% less fractional shortening and 5-9% slower contraction and 3-15% slower relaxation kinetics relative to their LV and IVS counterparts. Aided by a large dataset, we established relationships between contractile parameters and found contraction velocity, fractional shortening and relaxation velocity to be highly correlated. In conclusion, our findings are in line with contractile differences observed at the atrioventricular level, but can only partly explain the interventricular differences that exist at the organ level.

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-Pérot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Cardiac Microvascular Endothelial Enhancement of Cardiomyocyte Function Is Impaired by Inflammation and Restored by Empagliflozin.

The positive findings of the EMPA-REG OUTCOME trial (Randomized, Placebo-Controlled Cardiovascular Outcome Trial of Empagliflozin) on heart failure (HF) outcome in patients with type 2 diabetes mellitus suggest a direct effect of empagliflozin on the heart. These patients frequently have HF with preserved ejection fraction (HFpEF), in which a metabolic risk-related pro-inflammatory state induces cardiac microvascular endothelial cell (CMEC) dysfunction with subsequent cardiomyocyte (CM) contractility impairment. This study showed that CMECs confer a direct positive effect on contraction and relaxation of CMs, an effect that requires nitric oxide, is diminished after CMEC stimulation with tumor necrosis factor-α, and is restored by empagliflozin. Our findings on the effect of empagliflozin on CMEC-mediated preservation of CM function suggests that empagliflozin can be used to treat the cardiac mechanical implications of microvascular dysfunction in HFpEF.

End-diastolic force pre-activates cardiomyocytes and determines contractile force: role of titin and calcium.

Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca2+ ], titin-based stretch pre-activates cardiomyocytes during diastole and is a major determinant of force production in the subsequent contraction. Through this mechanism titin would play an important role in active force development and length-dependent activation. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) result in expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work loops that mimic the cardiac cycle in wild-type (WT) and heterozygous (HET) RBM20-deficient rats. In addition, we studied the role of diastolic [Ca2+ ] in membrane-permeabilized WT and HET cardiomyocytes. Intact cardiomyocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT) at low pacing frequencies, but this difference disappeared at high pacing frequencies. Length-dependent activation (force-sarcomere length relationship) was blunted in HET cardiomyocytes, but the force-end-diastolic force relationship was not different between HET and WT cardiomyocytes. To delineate the effects of diastolic [Ca2+ ] and titin pre-activation on force generation, measurements were performed in detergent-permeabilized cardiomyocytes. Cardiac twitches were simulated by transiently exposing permeabilized cardiomyocytes to 2 µm Ca2+ . Increasing diastolic [Ca2+ ] from 1 to 80 nm increased force development twofold in WT. Higher diastolic [Ca2+ ] was needed in HET. These findings are consistent with our hypothesis that pre-activation increases active force development. Highly compliant titin allows cells to function at higher diastolic [Ca2+ ].

High Fibroblast Growth Factor 23 concentrations in experimental renal failure impair calcium handling in cardiomyocytes.

The overwhelming majority of patients with chronic kidney disease (CKD) die prematurely before reaching end-stage renal disease, mainly due to cardiovascular causes, of which heart failure is the predominant clinical presentation. We hypothesized that CKD-induced increases of plasma FGF23 impair cardiac diastolic and systolic function. To test this, mice were subjected to 5/6 nephrectomy (5/6Nx) or were injected with FGF23 for seven consecutive days. Six weeks after surgery, plasma FGF23 was higher in 5/6Nx mice compared to sham mice (720 ± 31 vs. 256 ± 3 pg/mL, respectively, P = 0.034). In cardiomyocytes isolated from both 5/6Nx and FGF23 injected animals the rise of cytosolic calcium during systole was slowed (-13% and -19%, respectively) as was the decay of cytosolic calcium during diastole (-15% and -21%, respectively) compared to controls. Furthermore, both groups had similarly decreased peak cytosolic calcium content during systole. Despite lower cytosolic calcium contents in CKD or FGF23 pretreated animals, no changes were observed in contractile parameters of cardiomyocytes between the groups. Expression of calcium handling proteins and cardiac troponin I phosphorylation were similar between groups. Blood pressure, the heart weight:tibia length ratio, α-MHC/ß-MHC ratio and ANF mRNA expression, and systolic and diastolic function as measured by MRI did not differ between groups. In conclusion, the rapid, CKD-induced rise in plasma FGF23 and the similar decrease in cardiomyocyte calcium transients in modeled kidney disease and following 1-week treatment with FGF23 indicate that FGF23 partly mediates cardiomyocyte dysfunction in CKD.

Rapid frequency-dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes.

KEY POINTS: Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown. Rapid stimulation frequency-dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency. These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium. ABSTRACT: Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)-based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+ ]m ) was measured at different stimulation frequencies (0.1-4 Hz) and external calcium concentrations (1.8-3.6 mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura-4AM. The increases in [Ca2+ ]m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca2+ ]m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+ ]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium-calcium exchanger (mNCE) resulted in a rise in [Ca2+ ]m at baseline and, paradoxically, in an acceleration of Ca2+ release. IN CONCLUSION: rapid increases in [Ca2+ ]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat-to-beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering.

Mimicking the cardiac cycle in intact cardiomyocytes using diastolic and systolic force clamps; measuring power output.

AIMS: A single isolated cardiomyocyte is the smallest functional unit of the heart. Yet, all single isolated cardiomyocyte experiments have been limited by the lack of proper methods that could reproduce a physiological cardiac cycle. We aimed to investigate the contractile properties of a single cardiomyocyte that correctly mimic the cardiac cycle. METHODS AND RESULTS: By adjusting the parameters of the feedback loop, using a suitably engineered feedback system and recording the developed force and the length of a single rat cardiomyocyte during contraction and relaxation, we were able to construct force-length (FL) relations analogous to the pressure-volume (PV) relations at the whole heart level. From the cardiac loop graphs, we obtained, for the first time, the power generated by one single cardiomyocyte. CONCLUSION: Here, we introduce a new approach that by combining mechanics, electronics, and a new type optical force transducer can measure the FL relationship of a single isolated cardiomyocyte undergoing a mechanical loop that mimics the PV cycle of a beating heart.

Selective phosphorylation of PKA targets after ß-adrenergic receptor stimulation impairs myofilament function in Mybpc3-targeted HCM mouse model.

AIMS: Hypertrophic cardiomyopathy (HCM) has been associated with reduced ß-adrenergic receptor (ß-AR) signalling, leading downstream to a low protein kinase A (PKA)-mediated phosphorylation. It remained undefined whether all PKA targets will be affected similarly by diminished ß-AR signalling in HCM. We aimed to investigate the role of ß-AR signalling on regulating myofilament and calcium handling in an HCM mouse model harbouring a gene mutation (G > A transition on the last nucleotide of exon 6) in Mybpc3 encoding cardiac myosin-binding protein C. METHODS AND RESULTS: Cardiomyocyte contractile properties and phosphorylation state were measured in left ventricular permeabilized and intact cardiomyocytes isolated from heterozygous (HET) or homozygous (KI) Mybpc3-targeted knock-in mice. Significantly higher myofilament Ca²âºsensitivity and passive tension were detected in KI mice, which were normalized after PKA treatment. Loaded intact cardiomyocyte force-sarcomere length relation was impaired in both HET and KI mice, suggesting a reduced length-dependent activation. Unloaded cardiomyocyte function revealed an impaired myofilament contractile response to isoprenaline (ISO) in KI, whereas the calcium-handling response to ISO was maintained. This disparity was explained by an attenuated increase in cardiac troponin I (cTnI) phosphorylation in KI, whereas the increase in phospholamban (PLN) phosphorylation was maintained to wild-type values. CONCLUSION: These data provide evidence that in the KI HCM mouse model, ß-AR stimulation leads to preferential PKA phosphorylation of PLN over cTnI, resulting in an impaired inotropic and lusitropic response.

Decreased creatine kinase is linked to diastolic dysfunction in rats with right heart failure induced by pulmonary artery hypertension.

Our objective was to investigate the role of creatine kinase in the contractile dysfunction of right ventricular failure caused by pulmonary artery hypertension. Pulmonary artery hypertension and right ventricular failure were induced in rats by monocrotaline and compared to saline-injected control animals. In vivo right ventricular diastolic pressure-volume relationships were measured in anesthetized animals; diastolic force-length relationships in single enzymatically dissociated myocytes and myocardial creatine kinase levels by Western blot. We observed diastolic dysfunction in right ventricular failure indicated by significantly steeper diastolic pressure-volume relationships in vivo and diastolic force-length relationships in single myocytes. There was a significant reduction in creatine kinase protein expression in failing right ventricle. Dysfunction also manifested as a shorter diastolic sarcomere length in failing myocytes. This was associated with a Ca(2+)-independent mechanism that was sensitive to cross-bridge cycling inhibition. In saponin-skinned failing myocytes, addition of exogenous creatine kinase significantly lengthened sarcomeres, while in intact healthy myocytes, inhibition of creatine kinase significantly shortened sarcomeres. Creatine kinase inhibition also changed the relatively flat contraction amplitude-stimulation frequency relationship of healthy myocytes into a steeply negative, failing phenotype. Decreased creatine kinase expression leads to diastolic dysfunction. We propose that this is via local reduction in ATP:ADP ratio and thus to Ca(2+)-independent force production and diastolic sarcomere shortening. Creatine kinase inhibition also mimics a definitive characteristic of heart failure, the inability to respond to increased demand. Novel therapies for pulmonary artery hypertension are needed. Our data suggest that cardiac energetics would be a potential ventricular therapeutic target.

Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness.

Rapid changes in NADH and flavin autofluorescence in rat cardiac trabeculae reveal large mitochondrial complex II reserve capacity.

KEY POINTS: A photometry-based technique was developed to measure nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence and contractile properties simultaneously in intact rat trabeculae at a high time resolution. This provides insight into the function of mitochondrial complex I and II. Maximal complex I and complex II activities were determined in saponin-permeabilized right ventricular tissue by respirometry. In trabeculae, complex II function was considerably smaller than the maximal complex II activity, suggesting large complex II reserve capacity. Up-down asymmetry in NADH and FAD kinetics suggests a complex interaction between mitochondrial and contractile function. These data show that simultaneous measurement of contractile properties and NADH and FAD kinetics in cardiac trabeculae provides a mean to study the differences in complex I and II function in intact preparations in health and disease. ABSTRACT: The functional properties of cardiac mitochondria in intact preparations have been mainly studied by measurements of nicotinamide adenine dinucleotide (NADH) autofluorescence, which reflects mitochondrial complex I function. To assess complex II function, we extended this method by measuring flavin adenine dinucleotide (FAD)-related autofluorescence in electrically stimulated cardiac trabeculae isolated from the right ventricle from the rat at 27°C. NADH and FAD autofluorescence and tension responses were measured when stimulation frequency was increased from 0.5 Hz to 1, 2 or 3 Hz for 3 min, and thereafter decreased to 0.5 Hz. Maximal complex I and complex II activity in vitro were determined in saponin-permeabilized right ventricular tissue by respirometry. NADH responses upon an increase in stimulation frequency showed a rapid decline, followed by a slow recovery towards the initial level. FAD responses followed a similar time course, but in the opposite direction. The amplitudes of early rapid changes in the NADH and FAD concentration correlated well with the change in tension time integral per second (R(2)  = 0.833 and 0.660 for NADH and FAD, respectively), but with different slopes for the up and down transient. Maximal velocity of the increase in FAD concentration (16 ± 4 µm s(-1) ), measured upon an increase in stimulation frequency from 0.5 to 3 Hz was considerably smaller than that of the decrease in NADH (78 ± 13 µm s(-1) ). The respiration measurements indicated that the maximal velocity of NADH utilization (143 ± 14 µm s(-1) ) was 2 times smaller than that of FADH2 (291 ± 19 µm s(-1) ). This indicates that in cardiac mitochondria considerable complex II activity reserve is present.

Mouse intact cardiac myocyte mechanics: cross-bridge and titin-based stress in unactivated cells.

A carbon fiber-based cell attachment and force measurement system was used to measure the diastolic stress-sarcomere length (SL) relation of mouse intact cardiomyocytes, before and after the addition of actomyosin inhibitors (2,3-butanedione monoxime [BDM] or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% base length/second. Diastolic stress increased close to linear from 0 at SL 1.85 µm to 4.2 mN/mm(2) at SL 2.1 µm. The actomyosin inhibitors BDM and blebbistatin significantly lowered diastolic stress by ∼1.5 mN/mm(2) (at SL 2.1 µm, ∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. To test this further, calcium sensitivity of skinned myocytes was studied under conditions that simulate diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca(2+)] from below to above 100 nM. Mean active stress was significantly increased at [Ca(2+)] > 55 nM (pCa 7.25) and was ∼0.7 mN/mm(2) at 100 nM [Ca(2+)] (pCa 7.0) and ∼1.3 mN/mm(2) at 175 nM Ca(2+) (pCa 6.75). Inhibiting active stress in intact cells attached to carbon fibers at their resting SL and stretching the cells while first measuring restoring stress (pushing outward) and then passive stress (pulling inward) made it possible to determine the passive cell's mechanical slack SL as ∼1.95 µm and the restoring stiffness and passive stiffness of the cells around the slack SL each as ∼17 mN/mm(2)/µm/SL. Comparison between the results of intact and skinned cells shows that titin is the main contributor to restoring stress and passive stress of intact cells, but that under physiological conditions, calcium sensitivity is sufficiently high for actomyosin interaction to contribute to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.

Temporal pixel multiplexing for simultaneous high-speed, high-resolution imaging.

We introduce an imaging modality that, by offsetting pixel-exposure times during capture of a single image frame, embeds temporal information in each frame. This allows simultaneous acquisition of full-resolution images at native detector frame rates and high-speed image sequences at reduced resolution, without increasing bandwidth requirements. We demonstrate this method using macroscopic and microscopic examples, including imaging calcium transients in heart cells at 250 Hz using a 10-Hz megapixel camera.

A novel mutant cardiac troponin C disrupts molecular motions critical for calcium binding affinity and cardiomyocyte contractility.

Troponin C (TnC) belongs to the superfamily of EF-hand (helix-loop-helix) Ca(2+)-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca(2+)-binding Site II of TnC, TnC((E59D,D75Y)). Expression of recombinant TnC((E59D,D75Y)) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC(D75Y) was able to recapitulate the TnC((E59D,D75Y)) phenotype, whereas TnC(E59D) was functionally benign. Force-pCa relationships in TnC((E59D,D75Y)) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC((E59D,D75Y)) labeled with 2-[(4'-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca(2+) sensitivity and Ca(2+) binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca(2+)-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca(2+)-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.

Force-length relations in isolated intact cardiomyocytes subjected to dynamic changes in mechanical load.

We developed a dynamic force-length (FL) control system for single intact cardiomyocytes that uses a pair of compliant, computer-controlled, and piezo translator (PZT)-positioned carbon fibers (CF). CF are attached to opposite cell ends to afford dynamic and bidirectional control of the cell's mechanical environment. PZT and CF tip positions, as well as sarcomere length (SL), are simultaneously monitored in real time, and passive/active forces are calculated from CF bending. Cell force and length were dynamically adjusted by corresponding changes in PZT position, to achieve isometric, isotonic, or work-loop style contractions. Functionality of the technique was assessed by studying FL behavior of guinea pig intact cardiomyocytes. End-diastolic and end-systolic FL relations, obtained with varying preload and/or afterloads, were near linear, independent of the mode of contraction, and overlapping for the range of end-diastolic SLs tested (1.85-2.05 micro m). Instantaneous elastance curves, obtained from FL relation curves, showed an afterload-dependent decrease in time to peak elastance and slowed relaxation with both increased preload and afterload. The ability of the present system to independently and dynamically control preload, afterload, and transition between end-diastolic and end-systolic FL coordinates provides a valuable extension to the range of tools available for the study of single cardiomyocyte mechanics, to foster its interrelation with whole heart pathophysiology.

CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation.

Heart failure remains a leading cause of morbidity and mortality. The cellular mechanism underlying the development of cardiac dysfunction is a decrease in the number of viable cardiomyocytes. Recent observations have suggested that the adult heart may contain a progenitor cell population. Side population (SP) cells, characterized by a distinct Hoechst dye efflux pattern, have been shown to exist in multiple tissues and are capable of tissue-specific differentiation. In this report, we confirm the existence of a cardiac SP cell population, immunophenotypically distinct from bone marrow SP cells. Moreover, we demonstrate that among cardiac SP cells, the greatest potential for cardiomyogenic differentiation is restricted to cells negative for CD31 expression and positive for stem cell antigen 1 (Sca1) expression (CD31-/Sca1+). Furthermore, we determine that CD31-/Sca1+ cardiac SP cells are capable of both biochemical and functional cardiomyogenic differentiation into mature cardiomyocytes, with expression of cardiomyocyte-specific transcription factors and contractile proteins, as well as stimulated cellular contraction and intracellular calcium transients indistinguishable from adult cardiomyocytes. We also determine the necessity of cell-extrinsic signaling through coupling, although not fusion, with adult cardiomyocytes in regulating cardiomyogenic differentiation of cardiac SP cells. We, therefore, conclude that CD31-/Sca1+ cardiac SP cells represent a distinct cardiac progenitor cell population, capable of cardiomyogenic differentiation into mature cardiomyocytes through a process mediated by cellular coupling with adult cardiomyocytes.

Restoring force development by titin/connectin and assessment of Ig domain unfolding.

Titin/connectin is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is composed of serially-linked immunoglobulin (Ig)-like domains and several unique sequence elements. Here we address the role of titin/connectin in sarcomeres shortened to below the slack length (length attained by an un-activated cell in absence of external forces). Such shortened cells develop so-called restoring forces that re-extend the cells upon relaxation. The experiments that we present are based on a high throughput method with a rapid solution switching system which allows unattached single cardiac myocytes to be activated (resulting in shortening below the slack length) and then to be rapidly relaxed while their maximal re-lengthening velocity is measured at the sarcomere level (dSL/dtmax), with high-resolution imaging techniques. Experiments were carried out on myocytes that express different isoforms of titin/connectin. We measured the relation between dSL/dtmax and the minimal SL during contraction (SLmin) and determined the slope of this relation as a measure of 'restoring stiffness.' We found that the restoring stiffness correlates with the isoform expression profile with myocytes that express high levels of the stiff isoform (N2B) having the highest restoring stiffness. These results support the notion that titin/connectin is a bi-directional spring that develops passive force when stretched above the slack length and restoring force when shortened to below this length. We also discuss in detail the mechanisms that underlie titin/connectin's restoring force development and focus on whether or not unfolding of Ig domains plays a role.