Amir Landesberg

Improved vascular organization enhances functional integration of engineered skeletal muscle grafts

Severe traumatic events such as burns, and cancer therapy, often involve a significant loss of tissue, requiring surgical reconstruction by means of autologous muscle flaps. The scant availability of quality vascularized flaps and donor site morbidity often limit their use. Engineered vascularized grafts provide an alternative for this need. This work describes a first-time analysis, of the degree of in vitro vascularization and tissue organization, required to enhance the pace and efficacy of vascularized muscle graft integration in vivo. While one-day in vitro was sufficient for graft integration, a three-week culturing period, yielding semiorganized vessel structures and muscle fibers, significantly improved grafting efficacy. Implanted vessel networks were gradually replaced by host vessels, coupled with enhanced perfusion and capillary density. Upregulation of key graft angiogenic factors suggest its active role in promoting the angiogenic response. Transition from satellite cells to mature fibers was indicated by increased gene expression, increased capillary to fiber ratio, and similar morphology to normal muscle. We suggest a “relay” approach in which extended in vitro incubation, enabling the formation of a more structured vascular bed, allows for graft-host angiogenic collaboration that promotes anastomosis and vascular integration. The enhanced angiogenic response supports enhanced muscle regeneration, maturation, and integration.

Troponin elevation in severe sepsis and septic shock: The role of left ventricular diastolic dysfunction and right ventricular dilatation

Objective:Serum troponin concentrations predict mortality in almost every clinical setting they have been examined, including sepsis. However, the causes for troponin elevations in sepsis are poorly understood. We hypothesized that detailed investigation of myocardial dysfunction by echocardiography can provide insight into the possible causes of troponin elevation and its association with mortality in sepsis. Design:Prospective, analytic cohort study. Setting:Tertiary academic institute. Patients:A cohort of ICU patients with severe sepsis or septic shock. Interventions:Advanced echocardiography using global strain, strain-rate imaging and 3D left and right ventricular volume analyses in addition to the standard echocardiography, and concomitant high-sensitivity troponin-T measurement in patients with severe sepsis or septic shock. Measurements and Main Results:Two hundred twenty-five echocardiograms and concomitant high-sensitivity troponin-T measurements were performed in a cohort of 106 patients within the first days of severe sepsis or septic shock (2.1 ± 1.4 measurements/patient). Combining echocardiographic and clinical variables, left ventricular diastolic dysfunction defined as increased mitral E-to-strain-rate e′-wave ratio, right ventricular dilatation (increased right ventricular end-systolic volume index), high Acute Physiology and Chronic Health Evaluation-II score, and low glomerular filtration rate best correlated with elevated log-transformed concomitant high-sensitivity troponin-T concentrations (mixed linear model: t = 3.8, 3.3, 2.8, and –2.1 and p = 0.001, 0.0002, 0.006, and 0.007, respectively). Left ventricular systolic dysfunction determined by reduced strain-rate s′-wave or low ejection fraction did not significantly correlate with log(concomitant high-sensitivity troponin-T). Forty-one patients (39%) died in-hospital. Right ventricular end-systolic volume index and left ventricular strain-rate e′-wave predicted in-hospital mortality, independent of Acute Physiology and Chronic Health Evaluation-II score (logistic regression: Wald = 8.4, 6.6, and 9.8 and p = 0.004, 0.010, and 0.001, respectively). Concomitant high-sensitivity troponin-T predicted mortality in univariate analysis (Wald = 8.4; p = 0.004), but not when combined with right ventricular end-systolic volume index and strain-rate e′-wave in the multivariate analysis (Wald = 2.3, 4.6, and 6.2 and p = 0.13, 0.032, and 0.012, respectively). Conclusions:Left ventricular diastolic dysfunction and right ventricular dilatation are the echocardiographic variables correlating best with concomitant high-sensitivity troponin-T concentrations. Left ventricular diastolic and right ventricular systolic dysfunction seem to explain the association of troponin with mortality in severe sepsis and septic shock.

Sarcomere mechanics in uniform and non-uniform cardiac muscle: A link between pump function and arrhythmias

Starlings Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac muscle. These Ca2+ waves can reversibly be induced in muscle with non-uniform excitation contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). These observations suggest the unifying hypothesis that force feedback to Ca2+ binding by Tn-C is responsible for Starlings Law and the ESPVR in uniform myocardium and leads in non-uniform myocardium to a surge of Ca2+ released by the myofilaments during relaxation, which initiates arrhythmogenic propagating Ca2+ release by the SR.

Regulation of energy consumption in cardiac muscle: analysis of isometric contractions

The well-known linear relationship between oxygen consumption and force-length area or the force-time integral is analyzed here for isometric contractions. The analysis, which is based on a biochemical model that couples calcium kinetics with cross-bridge cycling, indicates that the change in the number of force-generating cross bridges with the change in the sarcomere length depends on the force generated by the cross bridges. This positive-feedback phenomenon is consistent with our reported cooperativity mechanism, whereby the affinity of the troponin for calcium and, hence, cross-bridge recruitment depends on the number of force-generating cross bridges. Moreover, it is demonstrated that a model that does not include a feedback mechanism cannot describe the dependence of energy consumption on the loading conditions. The cooperativity mechanism, which has been shown to determine the force-length relationship and the related Frank-Starling law, is shown here to provide the basis for the regulation of energy consumption in the cardiac muscle.The well-known linear relationship between oxygen consumption and force-length area or the force-time integral is analyzed here for isometric contractions. The analysis, which is based on a biochemical model that couples calcium kinetics with cross-bridge cycling, indicates that the change in the number of force-generating cross bridges with the change in the sarcomere length depends on the force generated by the cross bridges. This positive-feedback phenomenon is consistent with our reported cooperativity mechanism, whereby the affinity of the troponin for calcium and, hence, cross-bridge recruitment depends on the number of force-generating cross bridges. Moreover, it is demonstrated that a model that does not include a feedback mechanism cannot describe the dependence of energy consumption on the loading conditions. The cooperativity mechanism, which has been shown to determine the force-length relationship and the related Frank-Starling law, is shown here to provide the basis for the regulation of energy consumption in the cardiac muscle.

The sarcomeric control of energy conversion.

Abstract: The Frank‐Starling Law, Fenn Effect, and Sugas suggestions of cardiac muscle constant contractile efficiency establish the dependence of cardiac mechanics and energetics on the loading conditions. Consistent with these observations, this review suggests that the sarcomere control of contraction consists of two dominant feedbacks: (1) a cooperativity mechanism (positive feedback), whereby the number of force‐generating cross‐bridges (XBs) determines the affinity of calcium binding to the troponin regulatory protein; and (2) a mechanical (negative) feedback, whereby the filament shortening velocity affects the rate of XB turnover from the force to the non‐force generating conformation. The study explains the roles of these feedbacks in providing the adaptive control of energy consumption by the loading conditions and validates the dependence of the cooperativity mechanism on the number of strong XBs. The cooperativity mechanism regulates XB recruitment. It explains the cardiac force‐length calcium relationship, the related Frank‐Starling Law of the heart, and the adaptive control of new XB recruitment and the associated adenosine triphosphate (ATP) consumption. The mechanical feedback explains the force‐velocity relationship and the constant and high‐contractile efficiency. These mechanisms were validated by testing the force responses to large amplitude (100 nm/sarcomere) sarcomere length (SL) oscillations, in intact tetanized trabeculae (utilizing 30 μM cyclopiazonic). The force responses to large‐length oscillations lag behind the imposed oscillations at low extracellular calcium concentration ([Ca2+]0) and slow frequencies (<4 Hz, 25°C), yielding counterclockwise hystereses in the force‐length plane. The force was higher during shortening than during lengthening. The area within these hystereses corresponds to the external work generated from new XB recruitment during each oscillation, and it is determined by the delay in the force response. Characterization of the delayed response and its dependence on the SL, force, and calcium allows identification of the regulation of XB recruitment. The direct dependence of the phase on force indicates that XB recruitment is determined directly by the force (i.e., the number of strong XBs) and indirectly by SL or calcium. The suggested feedbacks determine cardiac energetics: 1) the constant and high contractile efficiency is an intrinsic property of the single XB, due to the mechanical feedback; and 2) the XBs are the myocyte sensors that modulate XB recruitment in response to length and load changes through the cooperativity mechanism.

Adaptive control of cardiac contraction to changes in loading: from theory of sarcomere dynamics to whole-heart function

The heart accommodates to rapid changes in demands. This review elucidates the adaptive control of cardiac function by loading conditions, and integrates the sarcomeric control of contraction (SCC) with isolated trabeculae and in vivo whole-heart studies. The SCC includes two feedback mechanisms: (1) cooperativity that regulates cross-bridge (XB) recruitment and the force–length relationship, and (2) mechanical feedback, whereby the filament-sliding velocity determines the XB-weakening rate and the force–velocity relationship. An isolated rat trabeculae study tested the suggested mechanisms during sarcomeric lengthening. The observations indicate that lengthening decreases the XB-weakening rate in a velocity-dependent manner, congruent with the suggested hypothesis and in contrast to alternative theories. A whole-heart level study in sheep reveals the existence of a preload-independent linear relationship between the external work (EW) and pressure–time integral during transient vena cava occlusions, for any given afterload, and not just at isovolumic contractions. The slope of this relationship decreases as the afterload increases. These findings highlight the mechanisms underlying the pressure (Frank’s phenomenon) and EW (Starling’s phenomenon) generation and the roles that the preload and afterload play. The theoretical, isolated fibers and whole-heart studies provide complementary information that strengthens our understanding of cardiac function from the top-down and bottom-up.

Stretch increases the force by decreasing cross-bridge weakening rate in the rat cardiac trabeculae

Stretch increases the force and decreases energy consumption in skeletal muscles. Cardiac muscle response to stretch has been scarcely investigated, and the underlying mechanisms remain elusive. We hypothesized that stretch increases the force by modulating the cross-bridge (XB) cycling rate. Trabeculae (n=10) were isolated from rat right ventricles. Sarcomere length was measured by laser diffraction and controlled by a fast servomotor. The number of strong XBs was assessed by measuring the dynamic stiffness. Ramp stretches at different velocities (V(SL) ≤ 2.17 μm/s) and onset times were imposed on sarcomeric isometric contractions. Stretches yielded identical increase in the stress and stiffness, implying that stretch increases force by increasing the number of XBs. A unique linear relationship was observed between the instantaneous normalized stress and stiffness for all the stretch velocities (1.01 ± 0.15, R(2)=0.98 ± 0.04), suggesting that the force per XB is constant for all stretch velocities. The increase in the stress during stretch normalized by the instantaneous isometric stress was denoted as the normalized stress enhancement (σ(E)). The normalized stiffness enhancement (K(E)) was defined accordingly. The rates of σ(E) and K(E) development depended linearly on the stretch velocity (7.06 ± 1.03 and 6.57 ± 1.17 μm(-1), respectively). Moreover, it was independent of the stretch onset time, indicating that it is not dominated by XB recruitment processes, since the number of available XBs and XB recruitment vary with time during the twitch. These observations strongly suggest that stretch decreases the rate of strong XB turnover to the weak conformation in a velocity-dependent manner.

Spatial Nonuniformity of Contraction Causes Arrhythmogenic Ca2+ Waves in Rat Cardiac Muscle

Abstract: Landesberg and Sidemans four state model of the cardiac cross‐bridge (XB) hypothesizes a feedback of force development to Ca2+ binding by troponin C (TnC). We have further modeled this behavior and observed that the force (F)‐Ca2+ relationship as well as the F‐sarcomere length (SL) relationship and the time course of F and Ca2+ transients in cardiac muscle can be reproduced faithfully by a single effect of F on deformation of the TnC‐Ca complex and, thereby, on the dissociation rate of Ca2+. Furthermore, this feedback predicts that rapid decline of F in the activated sarcomere causes release of Ca2+ from TnC‐Ca2+, which is sufficient to initiate arrhythmogenic Ca2+ release from the sarcoplasmic reticulum (SR). This work investigated the initiation of Ca2+ waves underlying triggered propagated contractions (TPCs) in rat cardiac trabeculae under conditions that simulate functional nonuniformity caused by mechanical or ischemic local damage of the myocardium. A mechanical discontinuity along the trabeculae was created by exposing the preparation to a small constant flow jet of solution that reduces excitation‐contraction coupling in myocytes within that segment. Force was measured, and SL as well as [Ca2+]i were measured regionally. When the jet contained caffeine, 2,3‐butanedione monoxime or low‐[Ca2+], muscle‐twitch F decreased and the sarcomeres in the exposed segment were stretched by shortening the normal regions outside the jet. During relaxation, the sarcomeres in the exposed segment shortened rapidly. Short trains of stimulation at 2.5 Hz reproducibly caused Ca2+ waves to rise from the borders exposed to the jet. Ca2+ waves started during F relaxation of the last stimulated twitch and propagated into segments both inside and outside of the jet. Arrhythmias, in the form of nondriven rhythmic activity, were triggered when the amplitude of the Ca2+ wave increased by raising [Ca2+]o. The arrhythmias disappeared when the muscle uniformity was restored by turning the jet off. These results show that nonuniform contraction can cause Ca2+ waves underlying TPCs, and suggest that Ca2+ dissociated from myofilaments plays an important role in the initiation of arrhythmogenic Ca2+ waves.

The external work-pressure time integral relationships and the afterload dependence of Frank-Starling mechanism.

The mechanisms underlying the Frank-Starling Law of the heart are elusive and the prevalent notion suggests that it is afterload independent. However, isolated fiber studies reveal that the afterload determines cardiac function through cross-bridge dependent mechanisms. The study explores the roles of the afterload, in situ. The LV was exposed by left-thoracotomy in adult sheep (72.6+/-8.2 kg, n=8). Pressure transducers were inserted into the LV and aorta, a flowmeter was placed around the aortic root, and the LV volume was assessed by sonocrystals. Occluders around the aorta and the inferior vena cava enabled control of the afterload and preload. Different afterloads were imposed by partial aortic occlusions. Transient inferior vena cava occlusions (IVCOs) were preformed whenever the afterload was steady. A highly linear relationship was found between the external work (EW) and pressure time integral (PTI) (R(2)=0.98+/-0.01) during each transient IVCO (n=48). The slope of the EW-PTI relationship (WPTiR) was preload independent since, for any given afterload, the EW and PTI lay on a straight line. Interestingly, the slope of the WPTiR was afterload dependant: The slope was 33.3+/-4.1 mJ/mmHg.s at baselines and decreased by 1.0+/-0.50 mJ/mmHg.s with every 1 mmHg.min/L increase in the peripheral resistance. A unique WPTiR was obtained during both the occlusion and release phases of each IVCO, while two distinct EW-preload or PTI-preload relationships were observed. The novel WPTiR ties the Frank (pressure development) and Starling (EW production) phenomena together. The dependence of the WPTiR on the afterload highlights the adaptive control of the Frank-Starling mechanisms to changes in the afterload.

The role of Ca2+ in coupling cardiac metabolism with regulation of contraction: In silico modeling

The heart adapts the rate of mitochondrial ATP production to energy demand without noticeable changes in the concentration of ATP, ADP and Pi, even for large transitions between different workloads. We suggest that the changes in demand modulate the cytosolic Ca2+ concentration that changes mitochondrial Ca2+ to regulate ATP production. Thus, the rate of ATP production by the mitochondria is coupled to the rate of ATP consumption by the sarcomere cross‐bridges (XBs). An integrated model was developed to couple cardiac metabolism and mitochondrial ATP production with the regulation of Ca2+ transient and ATP consumption by the sarcomere. The model includes two interrelated systems that run simultaneously utilizing two different integration steps: (1) The faster system describes the control of excitation contraction coupling with fast cytosolic Ca2+ transients, twitch mechanical contractions, and associated fluctuations in the mitochondrial Ca2+. (2) A slower system simulates the metabolic system, which consists of three different compartments: blood, cytosol, and mitochondria. The basic elements of the model are dynamic mass balances in the different compartments. Cytosolic Ca2+ handling is determined by four organelles: sarcolemmal Ca2+ influx and efflux; sarcoplasmic reticulum (SR) Ca2+ release and sequestration (SR); binding and dissociation from sarcomeric regulatory troponin complexes; and mitochondrial Ca2+ flows. Mitochondrial Ca2+ flows are determined by the Ca2+ uniporter and the mitochondrial Na+Ca2+ exchanger. The cytosolic Ca2+ determines the rate of ATP consumption by the sarcomere. Ca2+ binding to troponin regulates the rate of XBs recruitment and force development. The mitochondrial Ca2+ concentration determines the pyruvate dehydrogenase activity and the rate of ATP production by the F1‐F0 ATPase. The workload modulates the cytosolic Ca2+ concentration through feedback loops. The preload and afterload affect the number of strong XBs. The number of strong XBs determines the affinity of troponin for Ca2+, which alters the cytosolic Ca2+ transient. Model simulations quantify the role of Ca2+ in simultaneously controlling the power of contraction and the rate of ATP production. It explains the established empirical observation that significant changes in the metabolic fluxes can occur without significant changes in the key nucleotide (ATP and ADP) concentrations. Quantitative investigations of the mechanisms underlying the cardiac control of biochemical to mechanical energy conversion may lead to novel therapeutic modalities for the ischemic and failing myocardium.