Calibrating bead displacements in optical tweezers using acousto-optic deflectors

Displacements of optically trapped particles are often recorded using back-focal-plane interferometry. In order to calibrate the detector signals to displacements of the trapped object, several approaches are available. One often relies either on scanning a fixed bead across the waist of the laser beam or on analyzing the power spectrum of movements of the trapped bead. Here, we introduce an alternative method to perform this calibration. The method consists of very rapidly scanning the laser beam across the solvent-immersed, trapped bead using acousto-optic deflectors while recording the detector signals. It does not require any knowledge of solvent viscosity and bead diameter, and works in all types of samples, viscous or viscoelastic. Moreover, it is performed with the same bead as that used in the actual experiment. This represents marked advantages over established methods. © 2006 American Institute of Physics

Cardiomyocyte stiffness in diastolic heart failure

Background - Heart failure with preserved left ventricular (LV) ejection fraction (EF) is increasingly recognized and usually referred to as diastolic heart failure (DHF). Its pathogenetic mechanism remains unclear, partly because of a lack of myocardial biopsy material. Endomyocardial biopsy samples obtained from DHF patients were therefore analyzed for collagen volume fraction (CVF) and sarcomeric protein composition and compared with control samples. Single cardiomyocytes were isolated from these biopsy samples to assess cellular contractile performance. Methods and Results - DHF patients (n=12) had an LVEF of 71 ± 11%, an LV end-diastolic pressure (LVEDP) of 28±4 mm Hg, and no significant coronary artery stenoses. DHF patients had higher CVFs (7.5±4.0%, P<0.05) than did controls (n=8, 3.8±2.0%), and no conspicuous changes in sarcomeric protein composition were detected, Cardiomyocytes, mechanically isolated and treated with Triton X-100 to remove all membranes, were stretched to a sarcomere length of 2.2 μm and activated with solutions containing varying [Ca2+]. Compared with cardiomyocytes of controls, cardiomyocytes of DHF patients developed a similar total isometric force at maximal [Ca2+], but their resting tension (Fpassive) in the absence of Ca2+ was almost twice as high (6.6±3.0 versus 3.5±1.7 kN/m2, P<0.001). F passive and CVF combined yielded stronger correlations with LVEDP than did either alone. Administration of protein kinase A (PKA) to DHF cardiomyocytes lowered Fpassive to control values. Conclusions - DHF patients had stiffer cardiomyocytes, as evident from a higher F passive, at the same sarcomere length. Together with CVF, F passive determined in vivo diastolic LV dysfunction. Correction of this high Fpassive by PKA suggests that reduced phosphorylation of sarcomeric proteins is involved in DHF

Cardiac myosin-binding protein C mutations and hypertrophic cardiomyopathy haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction

Background-Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3(mut)). Methods and Results-Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c. 2373dupG, n=7; c. 2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3(mut). Protein expression of cMyBP-C was significantly reduced in MYBPC3(mut) by 33 +/- 5%. Cardiac MyBP-C phosphorylation in MYBPC3(mut) samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84 +/- 5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the beta-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3(mut) (20.2 +/- 2.7 kN/m(2)) compared with donor (34.5 +/- 1.1 kN/m(2)). Moreover, Ca2+ sensitivity was higher in MYBPC3(mut) (pCa(50)=5.62 +/- 0.04) than in donor (pCa(50)=5.54 +/- 0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic beta-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca2+ sensitivity between MYBPC3(mut) (pCa(50)=5.46 +/- 0.03) and donor (pCa(50)=5.48 +/- 0.02). Conclusions-Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca2+ sensitivity in MYBPC3(mut) is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction. (Circulation. 2009; 119: 1473-1483.

Diabetes mellitus worsens diastolic left ventricular dysfunction in aortic stenosis through altered myocardial structure and cardiomyocyte stiffness.

BACKGROUND: Aortic stenosis (AS) and diabetes mellitus (DM) are frequent comorbidities in aging populations. In heart failure, DM worsens diastolic left ventricular (LV) dysfunction, thereby adversely affecting symptoms and prognosis. Effects of DM on diastolic LV function were therefore assessed in aortic stenosis, and underlying myocardial mechanisms were identified. METHODS AND RESULTS: Patients referred for aortic valve replacement were subdivided into patients with AS and no DM (AS; n=46) and patients with AS and DM (AS-DM; n=16). Preoperative Doppler echocardiography and hemodynamics were implemented with perioperative LV biopsies. Histomorphometry and immunohistochemistry quantified myocardial collagen volume fraction and myocardial advanced glycation end product deposition. Isolated cardiomyocytes were stretched to 2.2-μm sarcomere length to measure resting tension (F(passive)). Expression and phosphorylation of titin isoforms were analyzed with gel electrophoresis with ProQ Diamond and SYPRO Ruby stains. Reduced LV end-diastolic distensibility in AS-DM was evident from higher LV end-diastolic pressure (21±1 mm Hg for AS versus 28±4 mm Hg for AS-DM; P=0.04) at comparable LV end-diastolic volume index and attributed to higher myocardial collagen volume fraction (AS, 12.9±1.1% versus AS-DM, 18.2±2.6%; P<0.001), more advanced glycation end product deposition in arterioles, venules, and capillaries (AS, 14.4±2.1 score per 1 mm(2) versus AS-DM, 31.4±6.1 score per 1 mm2; P=0.03), and higher F(passive) (AS, 3.5±1.7 kN/m2 versus AS-DM, 5.1±0.7 kN/m2; P=0.04). Significant hypophosphorylation of the stiff N2B titin isoform in AS-DM explained the higher F(passive) and normalization of F(passive) after in vitro treatment with protein kinase A. CONCLUSIONS: Worse diastolic LV dysfunction in AS-DM predisposes to heart failure and results from more myocardial fibrosis, more intramyocardial vascular advanced glycation end product deposition, and higher cardiomyocyte F(passive), which was related to hypophosphorylation of the N2B titin isoform

Pathomechanisms in heart failure: the contractile connection

Heart failure is a multi-factorial progressive disease in which eventually the contractile performance of the heart is insufficient to meet the demands of the body, even at rest. A distinction can be made on the basis of the cause of the disease in genetic and acquired heart failure and at the functional level between systolic and diastolic heart failure. Here the basic determinants of contractile function of myocardial cells will be reviewed and an attempt will be made to elucidate their role in the development of heart failure. The following topics are addressed: the tension generating capacity, passive tension, the rate of tension development, the rate of ATP utilisation, calcium sensitivity of tension development, phosphorylation of contractile proteins, length dependent activation and stretch activation. The reduction in contractile performance during systole can be attributed predominantly to a loss of cardiomyocytes (necrosis), myocyte disarray and a decrease in myofibrillar density all resulting in a reduction in the tension generating capacity and likely also to a mismatch between energy supply and demand of the myocardium. This leads to a decline in the ejection fraction of the heart. Diastolic dysfunction can be attributed to fibrosis and an increase in titin stiffness which result in an increase in stiffness of the ventricular wall and hampers the filling of the heart with blood during diastole. A large number of post translation modifications of regulatory sarcomeric proteins influence myocardial function by altering calcium sensitivity of tension development. It is still unclear whether in concert these influences are adaptive or maladaptive during the disease proces

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. 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 (R2 = 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

Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart.

Objective: The involvement of Calpain-I mediated proteolysis has been implicated in myofibrillar dysfunction of reperfused myocardium following ischemia (stunning). This study addresses the question whether ultrastructural alterations might be responsible for the depressed contractility. Methods: Mechanical properties and protein composition of isolated myocytes after Calpain-I exposure (1.25 U/ml; 10 min; 15°C; pCa 5.0) and of ischemic rat hearts following reperfusion were characterized. Results: Maximal isometric force (44±5 kN/m2) at pCa 4.5 (pCa = - log[Ca2+]) decreased by 42.5% in Triton permeabilized myocytes (n=11) after Calpain-I treatment. Force (and consequent myofilament disarrangement) during Calpain-I treatment was prevented by 40 mM BDM. The contractile force of Calpain-I exposed myocytes was significantly higher at submaximal levels of activation (pCa 5.5, 5.4 and 5.3) before maximal force development (pCa 4.5) than after maximal force development. The pCa50 value (5.40±0.02) determined from these initial test contractures did not differ significantly from that of untreated controls (5.44±0.03). However, after full activation Ca2+-sensitivity of force production in Calpain-I treated myocytes was significantly reduced (pCa50 5.34±0.02). This change in pCa50 was positively correlated with the reduction in maximal isometric force and was accompanied by sarcomere disorder. These findings imply that at least part of the Calpain-I induced mechanical alterations are dependent on force history. Measurements of the rate of force redevelopment after unloaded shortening suggested that Calpain-I did not affect cross-bridge kinetics. SDS gel electrophoresis and Western immunoblotting of Calpain-I treated myocytes revealed desmin degradation. The desmin content of postischemic myocardium was also reduced. Conclusion: Our results indicate that ultrastructural alterations may play an important role in the Calpain-I mediated cardiac dysfunction. (C) 2000 Elsevier Science B.V