Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.     

Power output is linearly related to MyHC content in rat skinned myocytes and isolated working hearts

The amount of work the heart can perform during ejection is governed by the inherent contractile properties of individual myocytes. One way to alter contractile properties is to alter contractile proteins such as myosin heavy chain (MyHC), which is known to demonstrate isoform plasticity in response to disease states. The purpose of this study was to examine myocyte functionality over the complete range of MyHC expression in heart, from 100% alpha-MyHC to 100% beta-MyHC, using euthyroid and hypothyroid rats. Peak power output in skinned cardiac myocytes decreased as a nearly linear function of beta-MyHC expression during maximal (r2 = 0.85, n = 44 myocyte preparations) and submaximal (r2 = 0.82, n = 31 myocyte preparations) Ca2+ activation. To determine whether single myocyte function translated to the level of the whole heart, power output was measured in working heart preparations expressing varied ratios of MyHC. Left ventricular power output of isolated working heart preparations also decreased as a linear function of increasing beta-MyHC expression (r2 = 0.82, n = 34 myocyte preparations). These results demonstrate that power output is highly dependent on MyHC expression in single myocytes, and this translates to the performance of working left ventricles.     

Caspase inhibition reduces cardiac myocyte dyshomeostasis and improves cardiac contractile function after major burn injury.

In the heart, thermal injury activates a group of intracellular cysteine proteases known as caspases, which have been suggested to contribute to myocyte inflammation and dyshomeostasis. In this study, Sprague-Dawley rats were given either a third-degree burn over 40% total body surface area plus conventional fluid resuscitation or sham burn injury. Experimental groups included 1) sham burn given vehicle, 400 microl DMSO; 2) sham burn given Q-VD-OPh (6 mg/kg), a highly specific and stable caspase inhibitor, 24 and 1 h prior to sham burn; 3) burn given vehicle, DMSO as above; 4) burn given Q-VD-OPh (6 mg/kg) 24 and 1 h prior to burn. Twenty-four hours postburn, hearts were harvested and studied with regard to myocardial intracellular sodium concentration, intracellular pH, ATP, and phosphocreatine (23Na/31P nuclear magnetic resonance); myocardial caspase-1, -3,and -8 expression; myocyte Na+ (fluorescent indicator, sodium-binding benzofurzan isophthalate); myocyte secretion of TNF-alpha, IL-1beta, IL-6, and IL-10; and myocardial performance (Langendorff). Burn injury treated with vehicle alone produced increased myocardial expression of caspase-1, -3, and -8, myocyte Na+ loading, cytokine secretion, and myocardial contractile depression; cellular pH, ATP, and phosphocreatine were stable. Q-VD-OPh treatment in burned rats attenuated myocardial caspase expression, prevented burn-related myocardial Na+ loading, attenuated myocyte cytokine responses, and improved myocardial contraction and relaxation. The present data suggest that signaling through myocardial caspases plays a pivotal role in burn-related myocyte sodium dyshomeostasis and myocyte inflammation, perhaps contributing to burn-related contractile dysfunction.     

Cardiac myocytes' dynamic contractile behavior differs depending on heart segment

Cardiac myocytes originating from different parts of the heart exhibit varying morphology and ultrastructure. However, the difference in their dynamic behavior is unclear. We examined the contraction of cardiac myocytes originating from the apex, ventricle, and atrium, and found that their dynamic behavior, such as amplitude and frequency of contraction, differs depending on the heart segment of origin. Using video microscopy and high‐precision image correlation, we found that: (1) apex myocytes exhibited the highest contraction rate (～17 beats/min); (2) ventricular myocytes exhibited the highest contraction amplitude (～5.2 micron); and (3) as myocyte contraction synchronized, their frequency did not change significantly, but the amplitude of contraction increased in apex and ventricular myocytes. In addition, as myocyte cultures mature they formed contractile filaments, further emphasizing the difference in myocyte dynamics is persistent. These results suggest that the dynamic behavior (in addition to static properties) of myocytes is dependent on their segment of origin. Biotechnol. Bioeng. 2013; 110: 628–636. © 2012 Wiley Periodicals, Inc.     

Simultaneous orientation and cellular force measurements in adult cardiac myocytes using three-dimensional polymeric microstructures

A number of techniques have been developed to monitor contractile function in isolated cardiac myocytes. While invaluable observations have been gained from these methodologies in understanding the contractile processes of the heart, they are invariably limited by their in vitro conditions. The present challenge is to develop innovative assays to mimic the in vivo milieu so as to allow a more physiological assessment of cardiac myocyte contractile forces. Here we demonstrate the use of a silicone elastomer, poly(dimethylsiloxane) (PDMS), to simultaneously orient adult cardiac myocytes in primary culture and measure the cellular forces in a three-dimensional substrate. The realignment of adult cardiac myocytes in long-term culture (7 days) was achieved due to directional reassembly of the myofibrils along the parallel polymeric sidewalls. The cellular mechanical forces were recorded in situ by observing the deformation of the micropillars embedded in the substrate. By coupling the cellular mechanical force measurements with on-chip cell orientation, this novel assay is expected to provide a means of a more physiological assessment of single cardiac myocyte contractile function and may facilitate the future development of in vitro assembled functional cardiac tissue.     

The sensitive giant: the role of titin-based stretch sensing complexes in the heart

Every heart beat is not equal. As physiological demands of the cardiovascular system change, cardiac myocytes modulate contractile parameters including the rate and force of contraction. Adaptive responses require the sensing of biomechanical signals involving the interface between the contractile cytoskeleton (myofibrils) and the sarcolemma at specialized cell-cell junctions (intercalated discs) and cell-substrate adhesion complexes (costameres). Recent studies have shed insight into how protein complexes within cardiac myocytes sense biomechanical signals, processes required for normal adaptive or pathological responses. This new evidence suggests that complexes associated with the giant, myofibrillar protein titin sense myocyte stretch. Here, we discuss evidence supporting titin being an ideal biomechanical sensor.     

Ablation of cardiac myosin–binding protein-C accelerates contractile kinetics in engineered cardiac tissue

Hypertrophic cardiomyopathy (HCM) caused by mutations in cardiac myosin–binding protein-C (cMyBP-C) is a heterogenous disease in which the phenotypic presentation is influenced by genetic, environmental, and developmental factors. Though mouse models have been used extensively to study the contractile effects of cMyBP-C ablation, early postnatal hypertrophic and dilatory remodeling may overshadow primary contractile defects. The use of a murine engineered cardiac tissue (mECT) model of cMyBP-C ablation in the present study permits delineation of the primary contractile kinetic abnormalities in an intact tissue model under mechanical loading conditions in the absence of confounding remodeling events. We generated mechanically integrated mECT using isolated postnatal day 1 mouse cardiac cells from both wild-type (WT) and cMyBP-C–null hearts. After culturing for 1 wk to establish coordinated spontaneous contraction, we measured twitch force and Ca²⁺ transients at 37°C during pacing at 6 and 9 Hz, with and without dobutamine. Compared with WT, the cMyBP-C–null mECT demonstrated faster late contraction kinetics and significantly faster early relaxation kinetics with no difference in Ca²⁺ transient kinetics. Strikingly, the ability of cMyBP-C–null mECT to increase contractile kinetics in response to adrenergic stimulation and increased pacing frequency were severely impaired. We conclude that cMyBP-C ablation results in constitutively accelerated contractile kinetics with preserved peak force with minimal contractile kinetic reserve. These functional abnormalities precede the development of the hypertrophic phenotype and do not result from alterations in Ca²⁺ transient kinetics, suggesting that alterations in contractile velocity may serve as the primary functional trigger for the development of hypertrophy in this model of HCM. Our findings strongly support a mechanism in which cMyBP-C functions as a physiological brake on contraction by positioning myosin heads away from the thin filament, a constraint which is removed upon adrenergic stimulation or cMyBP-C ablation.     

In vivo adenoviral transfer of sorcin reverses cardiac contractile abnormalities of diabetic cardiomyopathy

In many types of heart failure cardiac myocyte Ca(2+) handling is abnormal because of downregulation of key Ca(2+) - handling proteins like sarco(endo)plasmic reticulum Ca(2+) - ATPase (SERCA)2a and ryanodine receptor (RyR)2. The alteration in SERCA2a and RyR2 expression results in altered cytosolic Ca(2+) transients, leading to abnormal contraction. Sorcin is an EF-hand protein that confers the property of caffeine-activated intracellular Ca(2+) release in nonmuscle cells by interacting with RyR2. To determine whether sorcin could improve the contractile function of the heart, we overexpressed sorcin in the heart of either normal or diabetic mice and in adult rat cardiomyocytes with an adenoviral gene transfer approach. Sorcin overexpression was associated with an increase in cardiac contractility of the normal heart and dramatically rescued the abnormal contractile function of the diabetic heart. These effects could be attributed to an improvement of the Ca(2+) transients found in the cardiomyocyte after sorcin overexpression. Viral vector-mediated delivery of sorcin to cardiac myocytes is beneficial, resulting in improved contractile function in diabetic cardiomyopathy.     

Porcine cardiac myocyte power output is increased after chronic exercise training.

Chronic exercise training increases the functional capacity of the heart, perhaps by increased myocyte contractile function, as has been observed in rodent exercise models. We examined whether cardiac myocyte function is enhanced after chronic exercise training in Yucatan miniature swine, whose heart characteristics are similar to humans. Animals were designated as either sedentary (Sed), i.e., cage confined, or exercise trained (Ex), i.e., underwent 16-20 wk of progressive treadmill training. Exercise training efficacy was shown with significantly increased heart weight-to-body weight ratios, skeletal muscle citrate synthase activity, and exercise tolerance. Force-velocity properties were measured by attaching skinned cardiac myocytes between a force transducer and position motor, and shortening velocities were measured over a range of loads during maximal Ca2+ activation. Myocytes (n = 9) from nine Ex pigs had comparable force production but a approximately 30% increase in peak power output compared with myocytes (n = 8) from eight Sed. Interestingly, Ex myofibrillar samples also had higher baseline PKA-induced phosphorylation levels of cardiac troponin I, which may contribute to the increase in power. Overall, these results suggest that enhanced power-generating capacity of porcine cardiac myofibrils contributes to improved cardiac function after chronic exercise training.     

A novel method to study contraction characteristics of a single cardiac myocyte using carbon fibers

To facilitate cardiac muscle research, we developed a novel method by which the force and length of a single ventricular myocyte can be recorded with a pair of carbon graphite fibers attached firmly to both ends. One fiber was stiff, whereas the other fiber was compliant to allow the recording of force and shortening during twitch contractions. The image of the compliant carbon fiber was projected onto a pair of photodiodes, and their output was fed to a piezoelectric transducer after variable amplifications to alter the effective compliance of the carbon fiber. Thus contraction of the myocyte was induced under virtually isometric conditions as well as under auxotonic conditions. We obtained a bell-shaped relation between the compliance under an auxotonic load and the work output of the myocyte, which was directly related to myocyte performance in the heart. Because it is easy to attach myocytes to the experimental apparatus, the present method would allow us to study cardiac muscle mechanics at the cellular and molecular levels.     

Effects of Tityus serrulatus scorpion venom and one of its purified toxins (toxin gamma) on the isolated guinea-pig heart.

1. The effects of Tityus serrulatus scorpion venom and its most important toxin (toxin gamma) were investigated on isolated guinea-pig hearts, perfused with Locke solution, by the Langendorff's method. 2. The cardiac contraction, the coronary flow and the electrocardiogram (ECG) were simultaneously recorded. 3. Bolus injections of 25, 50 or 100 micrograms of scorpion venom and 2.5, 5 or 10 micrograms of toxin gamma in the heart evoked complex effects which were divided into 3 phases: an initial phase (tachycardia or bradycardia associated with an increase in contractile force), an intermediate phase (oscillations of cardiac rate, contractile force and coronary flow, due to wandering pacemakers) and a third phase (sinus tachycardia). 4. The bradycardia and the oscillations of rhythm were prevented by atropine, whereas the tachycardia and the increase in contractile force were prevented either by reserpine or propranolol. 5. Scorpion venom or toxin gamma induced a ST segment displacement in the ECG, explained by a transitory myocardial hypoxia, due to an increase in the contractile force and a simultaneous decrease of the coronary flow. 6. Perfusion of the heart with Locke solution containing 2% scorpion antivenom prevented almost totally the effects elicited by the venom. 7. It is concluded that the complex effects induced by scorpion venom and toxin gamma are due to the simultaneous release of acetylcholine and catecholamines from postganglionic nerve fibers in the heart.     

Control of stress propagation in the cytoplasm by prestress and loading frequency

One fundamental question in cell biology is how mechanical stresses are distributed inside the cytoplasm. Recently we have developed a synchronous detection approach to map cytoplasmic displacements and stresses using yellow fluorescent protein tagged mitochondria as fiducial markers of the cytoskeleton (CSK) in response to a localized load applied via an RGD-coated magnetic bead (7). We have shown that stresses are propagated to remote sites in the cytoplasm, a finding that contradicts continuum model predictions. Here we show that long distance force propagation in the cytoplasm was abolished when the contractile prestress in the CSK was lowered by relaxing agents and enhanced when the prestress was increased by contractile agonists. Surprisingly, when the loading frequency was varied from 0.03 Hz to 30 Hz, the total area of induced displacements (an index of the extent of stress propagation) first increased with loading frequency and then decreased with loading frequency. These results demonstrate that the long distance force propagation in living adherent cells might be controlled by the level of contractile prestress in the CSK and by the loading frequency.     

Single cell mechanics of rat cardiomyocytes under isometric, unloaded, and physiologically loaded conditions

One of the most salient characteristics of the heart is its ability to adjust work output to external load. To examine whether a single cardiomyocyte preparation retains this property, we measured the contractile function of a single rat cardiomyocyte under a wide range of loading conditions using a force-length measurement system implemented with adaptive control. A pair of carbon fibers was used to clamp the cardiomyocyte, attached to each end under a microscope. One fiber was stiff, serving as a mechanical anchor, while the bending motion of the compliant fiber was monitored for force-length measurement. Furthermore, by controlling the position of the compliant fiber using a piezoelectric translator based on adaptive control, we could change load dynamically during contractions. Under unloaded conditions, maximal shortening velocity was 106 +/- 8.9 microm/s (n = 13 cells), and, under isometric conditions, peak developed force reached 5,720 nN (41.6 +/- 5.6 mN/mm(2); n = 17 cells). When we simulated physiological working conditions consisting of an isometric contraction, followed by shortening and relaxation, the average work output was 828 +/- 123 J/m(3) (n = 20 cells). The top left corners of tension-length loops obtained under all of these conditions approximate a line, analogous to the end-systolic pressure-volume relation of the ventricle. All of the functional characteristics described were analogous to those established by studies using papillary muscle or trabeculae preparations. In conclusion, the present results confirmed the fact that each myocyte forms the functional basis for ventricular function and that single cell mechanics can be a link between subcellular events and ventricular mechanics.     

Blebbistatin specifically inhibits actin-myosin interaction in mouse cardiac muscle

Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3–2.8 µM). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type Ca²⁺ channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in Ca²⁺ sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 µM blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the Ca²⁺ influx and activation systems. heart; electrophysiology; permeabilized muscle     

Contractile parameters and occurrence of alternans in isolated rat myocardium at supra-physiological stimulation frequency

The cardiac refractory period prevents the heart from tetanic activation that is typically used in noncardiac striated muscle tissue. To what extent the refractory period prevents successive action potentials to activate the excitation-contraction coupling process and contractile machinery at supra-physiological rates, such as those present during ventricular fibrillation, is unknown. Using multicellular trabeculae isolated from rat hearts, we studied amplitude and kinetics of contraction at rates well above the normal in vivo rat heart range. We show that even at twice the maximal heart rate of the rat, little or no mechanical instability is observed; twitch contractions are at steady state, albeit with an elevated active diastolic force. Although the amplitude of contraction increased within in vivo heart rates (positive force-frequency response), at frequencies beyond the maximal heart rate (10-30 Hz) a steady decline of contractile amplitude is observed. Not until 30 Hz do the majority of the isolated muscle preparations show mechanical alternans, where strong and weak beats alternate. Interestingly, unlike striated limb skeletal muscle, fusing of twitch contractions did not cause a continuous increase in peak force: at frequencies of 10 Hz and above, systolic force declines with relatively little elevation in diastolic force. Contractile kinetics continued to accelerate, from 1 Hz up to 30 Hz, whereas the relative speed of contraction and relaxation remained closely coupled, reflected by a singular linear relationship between the maximal and minimal derivative of force (dF/dt). We conclude that cardiac muscle can produce mechanically stable steady-state contractions at supra-physiological pacing rates, while these contractions continue to decline in amplitude and increase in diastolic force past maximal heart rate.     

Metallothionein alleviates cardiac dysfunction in streptozotocin-induced diabetes: role of Ca2+ cycling proteins, NADPH oxidase, poly(ADP-Ribose) polymerase and myosin heavy chain isozyme

Diabetic cardiomyopathy contributes to high morbidity and mortality in diabetic populations. It is manifested by compromised ventricular contraction and prolonged relaxation attributable to multiple causative factors including oxidative stress. This study was designed to examine the effect of cardiac overexpression of the heavy metal scavenger metallothionein (MT) on cardiac contractile function, intracellular Ca(2+) cycling proteins, stress-activated signaling molecules and the myosin heavy chain (MHC) isozyme in diabetes. Adult male wild-type (FVB) and MT transgenic mice were made diabetic by a single injection of streptozotocin (STZ). Contractile properties were evaluated in cardiomyocytes including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dL/dt) and intracellular Ca(2+) fluorescence. Diabetes significantly depressed PS, +/-dL/dt, prolonged TPS, TR(90) and intracellular Ca(2+) clearing, elevated resting intracellular Ca(2+), reduced caffeine-induced sarcoplasmic reticulum Ca(2+) release and dampened stress tolerance at high stimulus frequencies. MT itself exhibited little effect on myocyte mechanics but it significantly alleviated STZ-induced myocyte contractile dysfunctions. Diabetes enhanced expression of the AT(1) receptor, phospholamban, the p47(phox) NADPH oxidase subunit and poly(ADP-ribose) polymerase (PARP), depressed the level of SERCA2a, Na(+)-Ca(2+) exchanger and triggered a beta-MHC isozyme switch. All of these STZ-induced alterations with the exception of depressed SERCA2a and enhanced phospholamban were reconciled by MT. Collectively, these data suggest a beneficial effect of MT in the therapeutics of diabetic cardiomyopathy, possibly through a mechanism related to NADPH oxidase, PARP and MHC isozyme switch.     

Coupling between contraction rhythm and calcium oscillation in a cultured cardiac myocyte: Fluctuation behaviour and its modelling

Isolated and cultured neonatal cardiac myocytes show self-sustaining cyclic contraction, and have the properties of a nonlinear oscillator. We study the dynamics of mechanical contraction and cellular free Ca²⁺ in a single myocyte for the purposes of gaining an insight into the way in which excitation and contraction processes are inter-related. The concentration of intracellular Ca²⁺ in the myocyte is also found to vary periodically associated with its rhythmic contraction. The Ca²⁺ dynamics maintains its self-oscillatory nature when the spontaneous contraction is abolished by pharmacological treatment using 2,3-butanedione monoxime. However, fluctuation analysis of the Ca²⁺ oscillation intervals reveals that there occurs a characteristic change in the fluctuation behaviour due to the suppression of contraction; the mean value and fluctuation magnitude of the oscillation intervals and the persistency of the fluctuation correlations at short timescales all increase after pharmacological treatment. We develop a new nonlinear model based on Bonhoeffer - van der Pol oscillators to elucidate the mechanisms behind the observed effects of cardiac contraction on the Ca²⁺ oscillation. The model is composed of three coupled nonlinear differential equations that can describe the dynamics of both excitation (cellular free Ca²⁺) and contraction. Almost all the experimental findings are successfully reproduced by adjusting a parameter in the model responsible for excitation - contraction coupling.     

Aging increases stiffness of cardiac myocytes measured by atomic force microscopy nanoindentation

It is well established that the aging heart exhibits left ventricular (LV) diastolic dysfunction and changes in mechanical properties, which are thought to be due to alterations in the extracellular matrix. We tested the hypothesis that the mechanical properties of cardiac myocytes significantly change with aging, which could contribute to the global changes in LV diastolic dysfunction. We used atomic force microscopy (AFM), which determines cellular mechanical property changes at nanoscale resolution in myocytes, from young (4 mo) and old (30 mo) male Fischer 344 x Brown Norway F1 hybrid rats. A measure of stiffness, i.e., apparent elastic modulus, was determined by analyzing the relationship between AFM indentation force and depth with the classical infinitesimal strain theory and by modeling the AFM probe as a blunted conical indenter. This is the first study to demonstrate a significant increase (P < 0.01) in the apparent elastic modulus of single, aging cardiac myocytes (from 35.1 +/- 0.7, n = 53, to 42.5 +/- 1.0 kPa, n = 58), supporting the novel concept that the mechanism mediating LV diastolic dysfunction in aging hearts resides, in part, at the level of the myocyte.     

Characterization and mechanism of P2X receptor-mediated increase in cardiac myocyte contractility

Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2X(4) receptor (P2X(4)R) is an important subunit of native cardiac P2X receptors. With transgenic mice with cardiac-specific overexpression of P2X(4)R (Tg) used as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and Ca(2+) transient effects and to determine the mechanism underlying the receptor-induced increase in myocyte contractility. In response to the agonist 2-methylthioATP (2-meSATP), Tg myocytes showed an increased intracellular Ca(2+) transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cAMP accumulation or L-type Ca(2+) channel activation. The increased Ca(2+) transient was not associated with any alteration in action potential duration, resting membrane potential, or diastolic fluorescence ratio or rates of rise and decline of the Ca(2+) transient. Simultaneous Ca(2+) transient and contraction measurements did not show any agonist-mediated change in myofilament Ca(2+) sensitivity. However, activation of the overexpressed P2X(4) receptor caused an enhanced SR Ca(2+) loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and Ca(2+) transient. Similar data were obtained in wild-type mouse ventricular myocytes. Thus an increased SR Ca(2+) content, occurring in the absence of cAMP accumulation or L-type Ca(2+) channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility.