The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A ... more The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A number of different titin isoforms with distinctly different structural elements within their central I-band region are expressed in human myocardium. Their coexpression has so far prevented an understanding of the respective contributions of the isoforms to myocardial elasticity. Using isoform-specific antibodies, we find in the present study that rat myocardium expresses predominantly the small N2B titin isoform, which allows us to characterize the elastic behavior of this isoform. The extensibility and force response of N2B titin were studied by using immunoelectron microscopy and by measuring the passive force-sarcomere length (SL) relation of single rat cardiac myocytes under a variety of mechanical conditions. Experimental results were compared with the predictions of a mechanical model in which the elastic titin segment behaves as two wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK segment (rich in proline [P], glutamate [E], valine [V], and lysine [K] residues), connected in series. The overall contour length was predicted from the sequence of N2B cardiac titin. According to mechanical measurements, above Ϸ2.2 m SL titin's elastic segment extends beyond its predicted contour length. Immunoelectron microscopy indicates that a prominent source of this contour-length gain is the extension of the unique N2B sequence (located between proximal tandem Ig segment and PEVK), and that Ig domain unfolding is negligible. Thus, the elastic region of N2B cardiac titin consists of three mechanically distinct extensible segments connected in series: the tandem Ig segment, the PEVK segment, and the unique N2B sequence. Rate-dependent and repetitive stretch-release experiments indicate that both the contour-length gain and the recovery from it involve kinetic processes, probably unfolding and refolding within the N2B segment. As a result, the contour length of titin's extensible segment depends on the rate and magnitude of the preceding mechanical perturbations. The rate of recovery from the length gain is slow, ensuring that the adjusted length is maintained through consecutive cardiac cycles and that hysteresis is minimal. Thus, as a result of the extensible properties of the unique N2B sequence, the I-band region of the N2B cardiac titin isoform functions as a molecular spring that is adjustable. (Circ Res.
Heart failure remains a leading cause of morbidity and mortality. The cellular mechanism underlyi... more 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 ...
Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated musc... more Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of ...
Titin is a giant protein spanning between the Z-and M-lines of the sarcomere. In the M-line the C... more Titin is a giant protein spanning between the Z-and M-lines of the sarcomere. In the M-line the C-terminal region of titin overlaps with that of oppositely oriented titin from the other half of the sarcomere. Furthermore, titin-binding proteins such as myomesin and M-protein localize in the M-line so as to form a complex. The M-line complex appears as a globular head-like structure in titin preparations. The exact structure and the molecular arrangement within this M-line complex is unknown. We analyzed the structure and stability of the M-line complex by using atomic force microscopy (AFM). We mechanically dissected the M-line complex of single surface-adsorbed titin oligomers by using AFM-based nanolithographic procedures. Titin was first deposited on mica so that the oligomers conformationally equilibrated on the surface.
Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control ... more Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca 2+ ], 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 [Ca 2+ ] 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 [Ca 2+ ] 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 Ca 2+. Increasing diastolic [Ca 2+ ] from 1 to 80 nM increased force development twofold in WT. Higher diastolic [Ca 2+ ] 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 [Ca 2+ ].
We developed a dynamic force-length (FL) control system for single intact cardiomyocytes that use... more 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 bi-directional 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.
This paper briefly recapitulates the Frank-Starling law of the heart, reviews approaches to estab... more This paper briefly recapitulates the Frank-Starling law of the heart, reviews approaches to establishing diastolic and systolic force-length behaviour in intact isolated cardiomyocytes, and introduces a dimensionless index called 'Frank-Starling Gain', calculated as the ratio of slopes of end-systolic and end-diastolic force-length relations. The benefits and limitations of this index are illustrated on the example of regional differences in Guinea pig intact ventricular cardiomyocyte mechanics. Potential applicability of the Frank-Starling Gain for the comparison of cell contractility changes upon stretch will be discussed in the context of intra-and inter-individual variability of cardiomyocyte properties.
We developed a dynamic force-length (FL) control system for single intact cardiomyocytes that use... more 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 bi-directional 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.
We introduce an imaging modality that, by offsetting pixel exposure times during capture of a sin... more We introduce an imaging modality that, by offsetting pixel exposure times during capture of a single image frame, embeds temporal information within 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. The method is demonstrated using macroscopic and microscopic examples, including imaging calcium transients in heart cells at 250Hz using a 10Hz megapixel camera.
A single isolated cardiomyocyte is the smallest functional unit of the heart. Yet, all single iso... more 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. 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 relations analogous to the pressure-volume relations at the whole heart level. From the cardiac loop graphs, we obtained, for the first time, the power generated by one single cardiomyocyte. Here, we introduce a new approach that by combining mechanics, electronics and a new type optical force transducer, can measure the force-length relationship of a single isolated cardiomyocyte undergoing a mechani...
Hypertrophic cardiomyopathy (HCM) has been associated with reduced β-adrenergic (β-AR) receptor s... more Hypertrophic cardiomyopathy (HCM) has been associated with reduced β-adrenergic (β-AR) receptor signaling, leading downstream to a low protein kinase A (PKA) mediated phosphorylation. It remained undefined whether all PKA targets will be affected similarly by diminished β-AR signaling in HCM. We aimed to investigate the role of β-AR signaling on regulating myofilament and calcium handling in an HCM mouse model harboring a gene mutation (G>A transition on the last nucleotide of exon 6) in Mybpc3 encoding cardiac myosin-binding protein C (cMyBP-C). Cardiomyocyte contractile properties as well as phosphorylation state were measured in left ventricular (LV) permeabilized and intact cardiomyocytes isolated from heterozygous (HET) or homozygous (KI) Mybpc3-targeted knock-in mice. A significant higher myofilament Ca(2+)-sensitivity and passive tension was detected in KI mice, which were normalized after PKA treatment. Loaded intact cardiomyocyte force-sarcomere length (SL) relation was ...
We introduce a imaging modality that works by transiently masking image-subregions during a singl... more We introduce a imaging modality that works by transiently masking image-subregions during a single exposure of a CCD frame. By offsetting subregion exposure time, temporal information is embedded within each stored frame, allowing simultaneous acquisition of a full high spatial resolution image and a high-speed image sequence without increasing bandwidth. The technique is demonstrated by imaging calcium transients in heart cells at 250 Hz with a 10 Hz megapixel camera.
The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A ... more The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A number of different titin isoforms with distinctly different structural elements within their central I-band region are expressed in human myocardium. Their coexpression has so far prevented an understanding of the respective contributions of the isoforms to myocardial elasticity. Using isoform-specific antibodies, we find in the present study that rat myocardium expresses predominantly the small N2B titin isoform, which allows us to characterize the elastic behavior of this isoform. The extensibility and force response of N2B titin were studied by using immunoelectron microscopy and by measuring the passive force-sarcomere length (SL) relation of single rat cardiac myocytes under a variety of mechanical conditions. Experimental results were compared with the predictions of a mechanical model in which the elastic titin segment behaves as two wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK segment (rich in proline [P], glutamate [E], valine [V], and lysine [K] residues), connected in series. The overall contour length was predicted from the sequence of N2B cardiac titin. According to mechanical measurements, above Ϸ2.2 m SL titin's elastic segment extends beyond its predicted contour length. Immunoelectron microscopy indicates that a prominent source of this contour-length gain is the extension of the unique N2B sequence (located between proximal tandem Ig segment and PEVK), and that Ig domain unfolding is negligible. Thus, the elastic region of N2B cardiac titin consists of three mechanically distinct extensible segments connected in series: the tandem Ig segment, the PEVK segment, and the unique N2B sequence. Rate-dependent and repetitive stretch-release experiments indicate that both the contour-length gain and the recovery from it involve kinetic processes, probably unfolding and refolding within the N2B segment. As a result, the contour length of titin's extensible segment depends on the rate and magnitude of the preceding mechanical perturbations. The rate of recovery from the length gain is slow, ensuring that the adjusted length is maintained through consecutive cardiac cycles and that hysteresis is minimal. Thus, as a result of the extensible properties of the unique N2B sequence, the I-band region of the N2B cardiac titin isoform functions as a molecular spring that is adjustable. (Circ Res.
Heart failure remains a leading cause of morbidity and mortality. The cellular mechanism underlyi... more 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 ...
Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated musc... more Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of ...
Titin is a giant protein spanning between the Z-and M-lines of the sarcomere. In the M-line the C... more Titin is a giant protein spanning between the Z-and M-lines of the sarcomere. In the M-line the C-terminal region of titin overlaps with that of oppositely oriented titin from the other half of the sarcomere. Furthermore, titin-binding proteins such as myomesin and M-protein localize in the M-line so as to form a complex. The M-line complex appears as a globular head-like structure in titin preparations. The exact structure and the molecular arrangement within this M-line complex is unknown. We analyzed the structure and stability of the M-line complex by using atomic force microscopy (AFM). We mechanically dissected the M-line complex of single surface-adsorbed titin oligomers by using AFM-based nanolithographic procedures. Titin was first deposited on mica so that the oligomers conformationally equilibrated on the surface.
Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control ... more Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca 2+ ], 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 [Ca 2+ ] 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 [Ca 2+ ] 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 Ca 2+. Increasing diastolic [Ca 2+ ] from 1 to 80 nM increased force development twofold in WT. Higher diastolic [Ca 2+ ] 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 [Ca 2+ ].
We developed a dynamic force-length (FL) control system for single intact cardiomyocytes that use... more 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 bi-directional 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.
This paper briefly recapitulates the Frank-Starling law of the heart, reviews approaches to estab... more This paper briefly recapitulates the Frank-Starling law of the heart, reviews approaches to establishing diastolic and systolic force-length behaviour in intact isolated cardiomyocytes, and introduces a dimensionless index called 'Frank-Starling Gain', calculated as the ratio of slopes of end-systolic and end-diastolic force-length relations. The benefits and limitations of this index are illustrated on the example of regional differences in Guinea pig intact ventricular cardiomyocyte mechanics. Potential applicability of the Frank-Starling Gain for the comparison of cell contractility changes upon stretch will be discussed in the context of intra-and inter-individual variability of cardiomyocyte properties.
We developed a dynamic force-length (FL) control system for single intact cardiomyocytes that use... more 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 bi-directional 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.
We introduce an imaging modality that, by offsetting pixel exposure times during capture of a sin... more We introduce an imaging modality that, by offsetting pixel exposure times during capture of a single image frame, embeds temporal information within 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. The method is demonstrated using macroscopic and microscopic examples, including imaging calcium transients in heart cells at 250Hz using a 10Hz megapixel camera.
A single isolated cardiomyocyte is the smallest functional unit of the heart. Yet, all single iso... more 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. 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 relations analogous to the pressure-volume relations at the whole heart level. From the cardiac loop graphs, we obtained, for the first time, the power generated by one single cardiomyocyte. Here, we introduce a new approach that by combining mechanics, electronics and a new type optical force transducer, can measure the force-length relationship of a single isolated cardiomyocyte undergoing a mechani...
Hypertrophic cardiomyopathy (HCM) has been associated with reduced β-adrenergic (β-AR) receptor s... more Hypertrophic cardiomyopathy (HCM) has been associated with reduced β-adrenergic (β-AR) receptor signaling, leading downstream to a low protein kinase A (PKA) mediated phosphorylation. It remained undefined whether all PKA targets will be affected similarly by diminished β-AR signaling in HCM. We aimed to investigate the role of β-AR signaling on regulating myofilament and calcium handling in an HCM mouse model harboring a gene mutation (G>A transition on the last nucleotide of exon 6) in Mybpc3 encoding cardiac myosin-binding protein C (cMyBP-C). Cardiomyocyte contractile properties as well as phosphorylation state were measured in left ventricular (LV) permeabilized and intact cardiomyocytes isolated from heterozygous (HET) or homozygous (KI) Mybpc3-targeted knock-in mice. A significant higher myofilament Ca(2+)-sensitivity and passive tension was detected in KI mice, which were normalized after PKA treatment. Loaded intact cardiomyocyte force-sarcomere length (SL) relation was ...
We introduce a imaging modality that works by transiently masking image-subregions during a singl... more We introduce a imaging modality that works by transiently masking image-subregions during a single exposure of a CCD frame. By offsetting subregion exposure time, temporal information is embedded within each stored frame, allowing simultaneous acquisition of a full high spatial resolution image and a high-speed image sequence without increasing bandwidth. The technique is demonstrated by imaging calcium transients in heart cells at 250 Hz with a 10 Hz megapixel camera.
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Papers by Michiel Helmes