Michiel Helmes

CD31− but Not CD31+ Cardiac Side Population Cells Exhibit Functional Cardiomyogenic Differentiation

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

Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes

Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 μmol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in ∼3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.

Titin Determines the Frank-Starling Relation in Early Diastole

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF–SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.

Temporal Pixel Multiplexing for simultaneous high-speed high-resolution imaging

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

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

A carbon fiber–based cell attachment and force measurement system was used to measure the diastolic stress–sarcomere length (SL) relation of mouse intact cardiomyocytes, before and after the addition of actomyosin inhibitors (2,3-butanedione monoxime [BDM] or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% base length/second. Diastolic stress increased close to linear from 0 at SL 1.85 µm to 4.2 mN/mm2 at SL 2.1 µm. The actomyosin inhibitors BDM and blebbistatin significantly lowered diastolic stress by ∼1.5 mN/mm2 (at SL 2.1 µm, ∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. To test this further, calcium sensitivity of skinned myocytes was studied under conditions that simulate diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca2+] from below to above 100 nM. Mean active stress was significantly increased at [Ca2+] > 55 nM (pCa 7.25) and was ∼0.7 mN/mm2 at 100 nM [Ca2+] (pCa 7.0) and ∼1.3 mN/mm2 at 175 nM Ca2+ (pCa 6.75). Inhibiting active stress in intact cells attached to carbon fibers at their resting SL and stretching the cells while first measuring restoring stress (pushing outward) and then passive stress (pulling inward) made it possible to determine the passive cell’s mechanical slack SL as ∼1.95 µm and the restoring stiffness and passive stiffness of the cells around the slack SL each as ∼17 mN/mm2/µm/SL. Comparison between the results of intact and skinned cells shows that titin is the main contributor to restoring stress and passive stress of intact cells, but that under physiological conditions, calcium sensitivity is sufficiently high for actomyosin interaction to contribute to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.

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

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

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

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

Synergistic role of ADP and Ca2+in diastolic myocardial stiffness: Cross-bridging the gap between energetics and Ca2+

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin‐related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca2+] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction.

The Power Output of Intact, Isolated Rat Cardiomyocytes

A method is presented to measure the power output of isolated intact cardiac myocytes. The mycoytes were attached to a very sensitive and fast force transducer on one side (OptiForce form Ionoptix) and a direct drive piezo-translator on the other side to stretch or shorten the myocyte. This allowed feedback based force control. Force clamps were used to impose a minimum ‘pre-load’ level and maximum ‘after-load’ level between which the myocyte was allowed to operate, mimicking the aortic and atrial pressure respectively. As long as the myocyte reaches the after load level in systole, the loaded shortening results in a force versus length curve that forms a work loop, analogous to the pressure volume curve in the whole heart. Within the measured range (0-1.5 µNewton pre-load, corresponding to sarcomere lengths of 1.86±0.07µm through 1.96±0.08µm) the end-systolic and end-diastolic force-length relations were linear. Measuring the area within each work loop provided the external work performed by the mycoyte for each contraction. A power curve was created by varying the after load for a given pre-load and multiplying the work per contraction with the pacing frequency. For rat myocytes at 37°C the peak power output increased linearly with pre-load within the range measured, and at a pre-load of 1.5 µNewton varied between 25 and 90 pJ.s-1. The large variation was mostly due to differences in cell size. Maximum power output was achieved at pacing frequencies between 6-8 Hz.

Mouse Intact and Skinned Cardiac Myocyte Mechanics: Crossbridge and Titin-Based Stress in Unactivated Cells

We investigated the contribution of actomyosin interactions in unactivated intact and skinned cardiomyocytes in physiologic conditions.A carbon fiber based cell-attachment system was used to measure the diastolic stress-sarcomere length (SL) relation of murine intact cardiomyocytes, before and after the addition of actomyosin inhibitors (BDM or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% length/s. Diastolic stress increased nearly linearly from 0 at SL 1.85 µm to 4.2 mN/mm2 at SL 2.1 µm. Actomyosin inhibitors lowered diastolic stress by ∼1.5 mN/mm2 at SL 2.1 µm (∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. Stretch-hold-release studies on skinned cardiomyocytes showed that as temperature changed from 24°C to 37°C, there was shortening of slack SL (from 1.90±0.01 μm to 1.89±0.01 μm) and increasing of both peak stress (∼35%) and steady state stress (∼26%). Shortening of slack SL and increasing stress could be inhibited by blebbistatin. This suggests that at physiologic temperature, crossbridge cycling takes place which contributes to diastolic stress. To extend this further, calcium sensitivity of skinned cardiomyocytes was studied under conditions that simulate physiologic diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca2+] from below to above 100 nM. Mean active stress was increased at [Ca2+] >55 nM (pCa 7.25) and was ∼0.7 mN/mm2 at 100 nM [Ca2+] (pCa 7.0) and ∼1.3 mN/mm2 at 175 nM [Ca2+] (pCa 6.75). The presence of active stress at pCa 7, which is a physiologic Ca2+ concentration of cytoplasm during diastole, confirms the contribution of crossbridge cycling to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.