C. Poggesi - Academia.edu (original) (raw)

Papers by C. Poggesi

Research paper thumbnail of Fisiologia - Molecole, cellule e sistemi - Volume II: Funzioni d’organo e integrazione sistemica

Questo libro nasce con l’intento di offrire agli studenti dei Corsi Universitari di carattere bio... more Questo libro nasce con l’intento di offrire agli studenti dei Corsi Universitari di carattere biomedico uno strumento di riferimento per lo studio dei meccanismi alla base del funzionamento degli esseri viventi, in particolare nel regno animale. La specificazione di “generale”, che compare nel titolo, indica che l’attenzione è principalmente rivolta ai processi fondamentali e unificanti dei fenomeni fisiologici, quelli che costituiscono le basi, di solito molecolari e cellulari, di funzioni fisiologiche anche apparentemente lontane tra loro. Poiché questo libro è destinato agli studenti di Corsi di Laurea differenti, dalle Scienze Biologiche alle Biotecnologie, ma anche di Scienze Naturali e Farmacia, si è ritenuto utile includere anche capitoli di Fisiologia dei sistemi. La sequenza degli argomenti si articola dal livello molecolare e cellulare, per considerare successivamente le interazioni fra le cellule e concludersi con la trattazione dei più importanti organi e sistemi d’organo

Research paper thumbnail of Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy

Proceedings of the National Academy of Sciences, 2017

Significance The homogenous propagation of the action potential in cardiac cells is guaranteed by... more Significance The homogenous propagation of the action potential in cardiac cells is guaranteed by a complex network of membrane invaginations called the T-tubular system. In cardiac diseases, T-tubules may show electrical defects that can compromise cell function. Here, we investigate the diffusional properties of fluorescent probes inside T-tubules to predict electrical conductivity of the tubular network. We apply this method to detecting alterations of T-tubule conductivity in a pathological setting characterized by compromised T-tubule integrity. We found that in heart failure, T-tubule conductivity is significantly reduced compared with healthy cardiac cells. A reduction in conductivity can impair the propagation of action potential across the network and may explain the presence of conduction defects found at the single tubular level.

Research paper thumbnail of Optogenetic manipulation of cardiac electrical dynamics using sub-threshold illumination: dissecting the role of cardiac alternans in terminating rapid rhythms

Cardiovascular Research

Funding Acknowledgements Type of funding sources: None. Cardiac action potential (AP) shape and p... more Funding Acknowledgements Type of funding sources: None. Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ions channel recovery and intracellular Ca2+-cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose a novel approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination) but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsins-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub...

Research paper thumbnail of P276Whole heart cytoarchitecture at sub-cellular resolution

Cardiovascular Research, 2018

Research paper thumbnail of Optogenetic Manipulation of Cardiac Electrical Dynamics Using Sub-Threshold Illumination: Dissecting the Role of Cardiac Alternans in Terminating Rapid Rhythms

SSRN Electronic Journal, 2021

Research paper thumbnail of Contractile properties of cardiomyocytes do not differ between obstructive and end-stage hypertrophic cardiomyopathy

Journal of Molecular and Cellular Cardiology, 2018

Background: One of the first clinically detectable alterations in heart function in hypertrophic ... more Background: One of the first clinically detectable alterations in heart function in hypertrophic cardiomyopathy (HCM) is a decline in diastolic function. Diastolic dysfunction is caused by changes in intrinsic properties of cardiomyocytes and/or an increase in fibrosis. We investigated if clinical and cellular parameters of diastolic function are sex-dependent in HCM. Methods and results: Cardiac tissue from the interventricular septum of HCM patients (26 female; 45 male) was obtained during myectomy preceded by echocardiography. At myectomy, female patients were older than male patients (50±15 vs 43±14 years; pb0.05). Female patients showed more advanced diastolic dysfunction than men evident from significantly higher values for E/e' ratio, LV filling pattern, TR velocity and left atrial diameter indexed for body surface. While most male patients (56%) showed mild (grade I) diastolic dysfunction, 50% of female patients showed grade III diastolic dysfunction. Passive tension in HCM cardiomyocytes was comparable to controls (1.93±0.09 vs 2.15±0.32), and myofilament calcium-sensitivity was higher in HCM compared to controls (pb0.0001), but no sex-differences were observed in myofilament function. In female HCM titin was more compliant (1.00±0.10 vs 0.74±0.04; pb0.05) and more fibrosis was present (6.42±0.95 vs 4.18±0.66; pb0.05) compared to males. Conclusion: Female HCM patients are older at time of myectomy and show greater impairment of diastolic function. Furthermore, LV and LA remodeling is increased in women when corrected for body surface area. At a cellular level HCM women showed increased titin compliance and a larger degree of interstitial fibrosis.

Research paper thumbnail of Myocardial overexpression of ANKRD1 causes sinus venosus defects and progressive diastolic dysfunction

Cardiovascular Research, 2019

AimsIncreased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have b... more AimsIncreased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have been associated to total anomalous pulmonary venous return and adult cardiomyopathy occurrence in humans. The link between increased ANKRD1 level and cardiac structural and functional disease is not understood. To get insight into this problem, we have generated a gain of function ANKRD1 mouse model by overexpressing ANKRD1 in the myocardium.Methods and resultsAnkrd1 is expressed non-homogeneously in the embryonic myocardium, with a dynamic nucleo-sarcomeric localization in developing cardiomyocytes. ANKRD1 transgenic mice present sinus venosus defect, which originates during development by impaired remodelling of early embryonic heart. Adult transgenic hearts develop diastolic dysfunction with preserved ejection fraction, which progressively evolves into heart failure, as shown histologically and haemodynamically. Transgenic cardiomyocyte structure, sarcomeric assembly, and stability are...

Research paper thumbnail of P1243Comparison of long-term clinical course and outcome of MYBPC3 - versus MYH7 - related hypertrophic cardiomyopathy

European Heart Journal, 2019

Introduction The presence of sarcomere mutations is a powerful predictor of heart failure-related... more Introduction The presence of sarcomere mutations is a powerful predictor of heart failure-related outcomes in Hypertrophic Cardiomyopathy (HCM). However, whether the prevalence of left ventricular (LV) dysfunction differs in patients with mutations in the two most prevalent HCM-associated genes (i.e. MYBPC3 and MYH7) is unclear. Purpose To ascertain lifetime trends in prevalence of LV dysfunction in HCM associated with pathogenic or likely-pathogenic MYBPC3 versus MYH7 mutations. Methods Clinical and instrumental records of 402 HCM patients with MYBPC3 (N=251) or MYH7 (N=151) mutations were retrospectively reviewed. Presence of systolic dysfunction (ejection fraction [EF] <50%) and diastolic dysfunction (Grade II and III) were assessed for each patient. In vitro analysis of septal myectomy samples was performed to further compare electro-mechanic properties of MYBC3 and MYH7 patients. Results Patients were diagnosed at a mean age of 39±17 years and 63% were men. At first evaluati...

Research paper thumbnail of Reply to Entcheva: The impact of T-tubules on action potential propagation in cardiac tissue

Proceedings of the National Academy of Sciences, 2018

Research paper thumbnail of Design of Biocompatible Liquid Cristal Elastomers Reproducing the Mechanical Properties of Human Cardiac Muscle

Biophysical Journal, 2019

Research paper thumbnail of Advanced Morpho-Functional Analysis on Ventricular and Atrial Tissue Reveals Cross-Bridge Kinetics Alterations and Sarcomere Energetic Impairment in Hcm Patients

Biophysical Journal, 2019

Research paper thumbnail of T-Tubular Electrical Defects Contribute to Blunted β-Adrenergic Response in Heart Failure

International journal of molecular sciences, Jan 3, 2016

Alterations of the β-adrenergic signalling, structural remodelling, and electrical failure of T-t... more Alterations of the β-adrenergic signalling, structural remodelling, and electrical failure of T-tubules are hallmarks of heart failure (HF). Here, we assess the effect of β-adrenoceptor activation on local Ca(2+) release in electrically coupled and uncoupled T-tubules in ventricular myocytes from HF rats. We employ an ultrafast random access multi-photon (RAMP) microscope to simultaneously record action potentials and Ca(2+) transients from multiple T-tubules in ventricular cardiomyocytes from a HF rat model of coronary ligation compared to sham-operated rats as a control. We confirmed that β-adrenergic stimulation increases the frequency of Ca(2+) sparks, reduces Ca(2+) transient variability, and hastens the decay of Ca(2+) transients: all these effects are similarly exerted by β-adrenergic stimulation in control and HF cardiomyocytes. Conversely, β-adrenergic stimulation in HF cells accelerates a Ca(2+) rise exclusively in the proximity of T-tubules that regularly conduct the acti...

Research paper thumbnail of Influence of some mechanical factors on inotropic level of left ventricular myocardium

Verhandlungen Der Deutschen Gesellschaft für Kreislaufforschung, 1974

Research paper thumbnail of The influence of temperature on the relaxation properties of rat papillary muscle

Bollettino della Società italiana di biologia sperimentale, Jan 15, 1982

Research paper thumbnail of P633 * Ranolazine reduces arrhythmogeneicity in transgenic mouse models of hypertrophic cardiomyopathy

Cardiovascular Research, 2014

Research paper thumbnail of Impaired Diastolic Function After Exchange of Endogenous Troponin I With C-Terminal Truncated Troponin I in Human Cardiac Muscle

Circulation Research, 2006

The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a k... more The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI 1–192 ) and full-length cTnI were expressed in Escherichia coli , purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 μm, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic β-adrenergic stimulatio...

Research paper thumbnail of Probing T-Tubular Electrophysiology by Random Access Two-Photon Microscopy in Cardiac Myocytes

Biophysical Journal, 2011

Research paper thumbnail of The Effect of Inorganic Phosphate on Force Generation in Single Myofibrils from Rabbit Skeletal Muscle

Biophysical Journal, 2000

In striated muscle, force generation and phosphate (P i) release are closely related. Alterations... more In striated muscle, force generation and phosphate (P i) release are closely related. Alterations in the [P i ] bathing skinned fibers have been used to probe key transitions of the mechanochemical coupling. Accuracy in this kind of studies is reduced, however, by diffusional barriers. A new perfusion technique is used to study the effect of [P i ] in single or very thin bundles (1-3 M in diameter; 5°C) of rabbit psoas myofibrils. With this technique, it is possible to rapidly jump [P i ] during contraction and observe the transient and steady-state effects on force of both an increase and a decrease in [P i ]. Steady-state isometric force decreases linearly with an increase in log[P i ] in the range 500 M to 10 mM (slope Ϫ0.4/decade). Between 5 and 200 M P i , the slope of the relation is smaller (ϳ Ϫ0.07/decade). The rate constant of force development (k TR) increases with an increase in [P i ] over the same concentration range. After rapid jumps in [P i ], the kinetics of both the force decrease with an increase in [P i ] (k Pi(ϩ)) and the force increase with a decrease in [P i ] (k Pi(Ϫ)) were measured. As observed in skinned fibers with caged P i , k Pi(ϩ) is about three to four times higher than k TR , strongly dependent on final [P i ], and scarcely modulated by the activation level. Unexpectedly, the kinetics of force increase after jumps from high to low [P i ] is slower: k Pi(Ϫ) is indistinguishable from k TR measured at the same [P i ] and has the same calcium sensitivity.

Research paper thumbnail of The HCM-Associated Cardiac Troponin T Mutation K280N Increases the Energetic Cost of Tension Generation in Human Cardiac Myofibrils

Biophysical Journal, 2013

We have used site-directed spin labeling and pulsed dipolar electron-electron paramagnetic resona... more We have used site-directed spin labeling and pulsed dipolar electron-electron paramagnetic resonance (DEER) to resolve the structure and dynamics of flexible and disordered regions of myosin binding protein-C (MyBP-C)'s cardiac isoform, with implications for the pathophysiology of hypertrophic cardiomyopathy (HCM). N-terminal domains of cMyBP-C contain binding domains for several interaction partners in the myofilament, including myosin heavy chain subfragment 2 (S2) and actin. We engineered pairs of labeling sites in protein fragments of mouse cMyBP-C to measure with high resolution distance and disorder between (1) domains C0 and C1, flanking the flexible Pro/Ala-rich linker, and between (2) domains C1 and C2, flanking the partially disordered phosphorylation motif, using DEER. Changes in distance and disorder were assessed for double-Cys mutant cMyBP-C's free in solution and when bound to myosin S2 or actin, with or without cMyBP-C phosphorylation by protein kinase A (PKA). Understanding conformational transitions in the flexible and dynamic portions of cMyBP-C upon actin-myosin binding and phosphorylation provide new molecular insight into defining its modulatory role in muscle force development.

Research paper thumbnail of Inulin uptake and washout in contracting and quiescent rat papillary muscle

Archives Internationales de Physiologie et de Biochimie, 1982

Equilibration and washout curves of inulin were determined in isolated rat papillary muscles. Inu... more Equilibration and washout curves of inulin were determined in isolated rat papillary muscles. Inulin uptake did not reach a steady-state value even after a long equilibration time. The effects of contractile activity were investigated. Isometric contractions increased the rate of inulin uptake and washout. In isotonic conditions, a smaller increase in the uptake and washout was observed. These changes could be attributed to an actual increase of inulin distribution space and/or to an accelerated diffusion kinetics.

Research paper thumbnail of Fisiologia - Molecole, cellule e sistemi - Volume II: Funzioni d’organo e integrazione sistemica

Questo libro nasce con l’intento di offrire agli studenti dei Corsi Universitari di carattere bio... more Questo libro nasce con l’intento di offrire agli studenti dei Corsi Universitari di carattere biomedico uno strumento di riferimento per lo studio dei meccanismi alla base del funzionamento degli esseri viventi, in particolare nel regno animale. La specificazione di “generale”, che compare nel titolo, indica che l’attenzione è principalmente rivolta ai processi fondamentali e unificanti dei fenomeni fisiologici, quelli che costituiscono le basi, di solito molecolari e cellulari, di funzioni fisiologiche anche apparentemente lontane tra loro. Poiché questo libro è destinato agli studenti di Corsi di Laurea differenti, dalle Scienze Biologiche alle Biotecnologie, ma anche di Scienze Naturali e Farmacia, si è ritenuto utile includere anche capitoli di Fisiologia dei sistemi. La sequenza degli argomenti si articola dal livello molecolare e cellulare, per considerare successivamente le interazioni fra le cellule e concludersi con la trattazione dei più importanti organi e sistemi d’organo

Research paper thumbnail of Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy

Proceedings of the National Academy of Sciences, 2017

Significance The homogenous propagation of the action potential in cardiac cells is guaranteed by... more Significance The homogenous propagation of the action potential in cardiac cells is guaranteed by a complex network of membrane invaginations called the T-tubular system. In cardiac diseases, T-tubules may show electrical defects that can compromise cell function. Here, we investigate the diffusional properties of fluorescent probes inside T-tubules to predict electrical conductivity of the tubular network. We apply this method to detecting alterations of T-tubule conductivity in a pathological setting characterized by compromised T-tubule integrity. We found that in heart failure, T-tubule conductivity is significantly reduced compared with healthy cardiac cells. A reduction in conductivity can impair the propagation of action potential across the network and may explain the presence of conduction defects found at the single tubular level.

Research paper thumbnail of Optogenetic manipulation of cardiac electrical dynamics using sub-threshold illumination: dissecting the role of cardiac alternans in terminating rapid rhythms

Cardiovascular Research

Funding Acknowledgements Type of funding sources: None. Cardiac action potential (AP) shape and p... more Funding Acknowledgements Type of funding sources: None. Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ions channel recovery and intracellular Ca2+-cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose a novel approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination) but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsins-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub...

Research paper thumbnail of P276Whole heart cytoarchitecture at sub-cellular resolution

Cardiovascular Research, 2018

Research paper thumbnail of Optogenetic Manipulation of Cardiac Electrical Dynamics Using Sub-Threshold Illumination: Dissecting the Role of Cardiac Alternans in Terminating Rapid Rhythms

SSRN Electronic Journal, 2021

Research paper thumbnail of Contractile properties of cardiomyocytes do not differ between obstructive and end-stage hypertrophic cardiomyopathy

Journal of Molecular and Cellular Cardiology, 2018

Background: One of the first clinically detectable alterations in heart function in hypertrophic ... more Background: One of the first clinically detectable alterations in heart function in hypertrophic cardiomyopathy (HCM) is a decline in diastolic function. Diastolic dysfunction is caused by changes in intrinsic properties of cardiomyocytes and/or an increase in fibrosis. We investigated if clinical and cellular parameters of diastolic function are sex-dependent in HCM. Methods and results: Cardiac tissue from the interventricular septum of HCM patients (26 female; 45 male) was obtained during myectomy preceded by echocardiography. At myectomy, female patients were older than male patients (50±15 vs 43±14 years; pb0.05). Female patients showed more advanced diastolic dysfunction than men evident from significantly higher values for E/e' ratio, LV filling pattern, TR velocity and left atrial diameter indexed for body surface. While most male patients (56%) showed mild (grade I) diastolic dysfunction, 50% of female patients showed grade III diastolic dysfunction. Passive tension in HCM cardiomyocytes was comparable to controls (1.93±0.09 vs 2.15±0.32), and myofilament calcium-sensitivity was higher in HCM compared to controls (pb0.0001), but no sex-differences were observed in myofilament function. In female HCM titin was more compliant (1.00±0.10 vs 0.74±0.04; pb0.05) and more fibrosis was present (6.42±0.95 vs 4.18±0.66; pb0.05) compared to males. Conclusion: Female HCM patients are older at time of myectomy and show greater impairment of diastolic function. Furthermore, LV and LA remodeling is increased in women when corrected for body surface area. At a cellular level HCM women showed increased titin compliance and a larger degree of interstitial fibrosis.

Research paper thumbnail of Myocardial overexpression of ANKRD1 causes sinus venosus defects and progressive diastolic dysfunction

Cardiovascular Research, 2019

AimsIncreased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have b... more AimsIncreased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have been associated to total anomalous pulmonary venous return and adult cardiomyopathy occurrence in humans. The link between increased ANKRD1 level and cardiac structural and functional disease is not understood. To get insight into this problem, we have generated a gain of function ANKRD1 mouse model by overexpressing ANKRD1 in the myocardium.Methods and resultsAnkrd1 is expressed non-homogeneously in the embryonic myocardium, with a dynamic nucleo-sarcomeric localization in developing cardiomyocytes. ANKRD1 transgenic mice present sinus venosus defect, which originates during development by impaired remodelling of early embryonic heart. Adult transgenic hearts develop diastolic dysfunction with preserved ejection fraction, which progressively evolves into heart failure, as shown histologically and haemodynamically. Transgenic cardiomyocyte structure, sarcomeric assembly, and stability are...

Research paper thumbnail of P1243Comparison of long-term clinical course and outcome of MYBPC3 - versus MYH7 - related hypertrophic cardiomyopathy

European Heart Journal, 2019

Introduction The presence of sarcomere mutations is a powerful predictor of heart failure-related... more Introduction The presence of sarcomere mutations is a powerful predictor of heart failure-related outcomes in Hypertrophic Cardiomyopathy (HCM). However, whether the prevalence of left ventricular (LV) dysfunction differs in patients with mutations in the two most prevalent HCM-associated genes (i.e. MYBPC3 and MYH7) is unclear. Purpose To ascertain lifetime trends in prevalence of LV dysfunction in HCM associated with pathogenic or likely-pathogenic MYBPC3 versus MYH7 mutations. Methods Clinical and instrumental records of 402 HCM patients with MYBPC3 (N=251) or MYH7 (N=151) mutations were retrospectively reviewed. Presence of systolic dysfunction (ejection fraction [EF] <50%) and diastolic dysfunction (Grade II and III) were assessed for each patient. In vitro analysis of septal myectomy samples was performed to further compare electro-mechanic properties of MYBC3 and MYH7 patients. Results Patients were diagnosed at a mean age of 39±17 years and 63% were men. At first evaluati...

Research paper thumbnail of Reply to Entcheva: The impact of T-tubules on action potential propagation in cardiac tissue

Proceedings of the National Academy of Sciences, 2018

Research paper thumbnail of Design of Biocompatible Liquid Cristal Elastomers Reproducing the Mechanical Properties of Human Cardiac Muscle

Biophysical Journal, 2019

Research paper thumbnail of Advanced Morpho-Functional Analysis on Ventricular and Atrial Tissue Reveals Cross-Bridge Kinetics Alterations and Sarcomere Energetic Impairment in Hcm Patients

Biophysical Journal, 2019

Research paper thumbnail of T-Tubular Electrical Defects Contribute to Blunted β-Adrenergic Response in Heart Failure

International journal of molecular sciences, Jan 3, 2016

Alterations of the β-adrenergic signalling, structural remodelling, and electrical failure of T-t... more Alterations of the β-adrenergic signalling, structural remodelling, and electrical failure of T-tubules are hallmarks of heart failure (HF). Here, we assess the effect of β-adrenoceptor activation on local Ca(2+) release in electrically coupled and uncoupled T-tubules in ventricular myocytes from HF rats. We employ an ultrafast random access multi-photon (RAMP) microscope to simultaneously record action potentials and Ca(2+) transients from multiple T-tubules in ventricular cardiomyocytes from a HF rat model of coronary ligation compared to sham-operated rats as a control. We confirmed that β-adrenergic stimulation increases the frequency of Ca(2+) sparks, reduces Ca(2+) transient variability, and hastens the decay of Ca(2+) transients: all these effects are similarly exerted by β-adrenergic stimulation in control and HF cardiomyocytes. Conversely, β-adrenergic stimulation in HF cells accelerates a Ca(2+) rise exclusively in the proximity of T-tubules that regularly conduct the acti...

Research paper thumbnail of Influence of some mechanical factors on inotropic level of left ventricular myocardium

Verhandlungen Der Deutschen Gesellschaft für Kreislaufforschung, 1974

Research paper thumbnail of The influence of temperature on the relaxation properties of rat papillary muscle

Bollettino della Società italiana di biologia sperimentale, Jan 15, 1982

Research paper thumbnail of P633 * Ranolazine reduces arrhythmogeneicity in transgenic mouse models of hypertrophic cardiomyopathy

Cardiovascular Research, 2014

Research paper thumbnail of Impaired Diastolic Function After Exchange of Endogenous Troponin I With C-Terminal Truncated Troponin I in Human Cardiac Muscle

Circulation Research, 2006

The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a k... more The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI 1–192 ) and full-length cTnI were expressed in Escherichia coli , purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 μm, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic β-adrenergic stimulatio...

Research paper thumbnail of Probing T-Tubular Electrophysiology by Random Access Two-Photon Microscopy in Cardiac Myocytes

Biophysical Journal, 2011

Research paper thumbnail of The Effect of Inorganic Phosphate on Force Generation in Single Myofibrils from Rabbit Skeletal Muscle

Biophysical Journal, 2000

In striated muscle, force generation and phosphate (P i) release are closely related. Alterations... more In striated muscle, force generation and phosphate (P i) release are closely related. Alterations in the [P i ] bathing skinned fibers have been used to probe key transitions of the mechanochemical coupling. Accuracy in this kind of studies is reduced, however, by diffusional barriers. A new perfusion technique is used to study the effect of [P i ] in single or very thin bundles (1-3 M in diameter; 5°C) of rabbit psoas myofibrils. With this technique, it is possible to rapidly jump [P i ] during contraction and observe the transient and steady-state effects on force of both an increase and a decrease in [P i ]. Steady-state isometric force decreases linearly with an increase in log[P i ] in the range 500 M to 10 mM (slope Ϫ0.4/decade). Between 5 and 200 M P i , the slope of the relation is smaller (ϳ Ϫ0.07/decade). The rate constant of force development (k TR) increases with an increase in [P i ] over the same concentration range. After rapid jumps in [P i ], the kinetics of both the force decrease with an increase in [P i ] (k Pi(ϩ)) and the force increase with a decrease in [P i ] (k Pi(Ϫ)) were measured. As observed in skinned fibers with caged P i , k Pi(ϩ) is about three to four times higher than k TR , strongly dependent on final [P i ], and scarcely modulated by the activation level. Unexpectedly, the kinetics of force increase after jumps from high to low [P i ] is slower: k Pi(Ϫ) is indistinguishable from k TR measured at the same [P i ] and has the same calcium sensitivity.

Research paper thumbnail of The HCM-Associated Cardiac Troponin T Mutation K280N Increases the Energetic Cost of Tension Generation in Human Cardiac Myofibrils

Biophysical Journal, 2013

We have used site-directed spin labeling and pulsed dipolar electron-electron paramagnetic resona... more We have used site-directed spin labeling and pulsed dipolar electron-electron paramagnetic resonance (DEER) to resolve the structure and dynamics of flexible and disordered regions of myosin binding protein-C (MyBP-C)'s cardiac isoform, with implications for the pathophysiology of hypertrophic cardiomyopathy (HCM). N-terminal domains of cMyBP-C contain binding domains for several interaction partners in the myofilament, including myosin heavy chain subfragment 2 (S2) and actin. We engineered pairs of labeling sites in protein fragments of mouse cMyBP-C to measure with high resolution distance and disorder between (1) domains C0 and C1, flanking the flexible Pro/Ala-rich linker, and between (2) domains C1 and C2, flanking the partially disordered phosphorylation motif, using DEER. Changes in distance and disorder were assessed for double-Cys mutant cMyBP-C's free in solution and when bound to myosin S2 or actin, with or without cMyBP-C phosphorylation by protein kinase A (PKA). Understanding conformational transitions in the flexible and dynamic portions of cMyBP-C upon actin-myosin binding and phosphorylation provide new molecular insight into defining its modulatory role in muscle force development.

Research paper thumbnail of Inulin uptake and washout in contracting and quiescent rat papillary muscle

Archives Internationales de Physiologie et de Biochimie, 1982

Equilibration and washout curves of inulin were determined in isolated rat papillary muscles. Inu... more Equilibration and washout curves of inulin were determined in isolated rat papillary muscles. Inulin uptake did not reach a steady-state value even after a long equilibration time. The effects of contractile activity were investigated. Isometric contractions increased the rate of inulin uptake and washout. In isotonic conditions, a smaller increase in the uptake and washout was observed. These changes could be attributed to an actual increase of inulin distribution space and/or to an accelerated diffusion kinetics.