While the reductionist approach has been fruitful in understanding the molecular basis of muscle function, intact excitable muscle preparations are still important as experimental model systems. We present here methods that are useful for preparing cardiac papillary muscle and cardiac slices, which represent macroscopic experimental model systems with fully intact intercellular and intracellular structures. The maintenance of these in vivo structures for experimentation in vitro have made these model systems especially useful for testing the functional effects of protein mutations and pharmaceutical candidates. We provide solutions recipes for dissection and recording, instructions for removing and preparing the cardiac papillary muscles, as well as instruction for preparing cardiac slices. These instructions are suitable for beginning experimentalists but may be useful for veteran muscle physiologists hoping to reacquaint themselves with macroscopic functional analyses.

Patients suffering from heart failure and left bundle branch block show electrical ventricular dyssynchrony causing an abnormal blood pumping. Cardiac resynchronization therapy (CRT) is recommended for these patients. Patients with positive therapy response normally present QRS shortening and an increased left ventricle (LV) ejection fraction. However, around one third do not respond favorably. Therefore, optimal location of pacing leads, timing delays between leads and/or choosing related biomarkers is crucial to achieve the best possible degree of ventricular synchrony during CRT application. In this study, computational modeling is used to predict the optimal location and delay of pacing leads to improve CRT response. We use a 3D electrophysiological computational model of the heart and torso to get insight into the changes in the activation patterns obtained when the heart is paced from different regions and for different atrioventricular and interventricular delays. The model represents a heart with left bundle branch block and heart failure, and allows a detailed and accurate analysis of the electrical changes observed simultaneously in the myocardium and in the QRS complex computed in the precordial leads. Computational simulations were performed using a modified version of the O'Hara et al. action potential model, the most recent mathematical model developed for human ventricular electrophysiology. The optimal location for the pacing leads was determined by QRS maximal reduction. Additionally, the influence of Purkinje system on CRT response was assessed and correlation analysis between several parameters of the QRS was made. Simulation results showed that the right ventricle (RV) upper septum near the outflow tract is an alternative location to the RV apical lead. Furthermore, LV endocardial pacing provided better results as compared to epicardial stimulation. Finally, the time to reach the 90% of the QRS area was a good predictor of the instant at which 90% of the ventricular tissue was activated. Thus, the time to reach the 90% of the QRS area is suggested as an additional index to assess CRT effectiveness to improve biventricular synchrony.

Introduction: Management of acute myocardial infarction (MI) mandates careful optimization of volemia, which can be challenging due to the inherent risk of congestion. Increased myocardial compliance in response to stretching, known as stretch-induced compliance (SIC), has been recently characterized and partly ascribed to cGMP/cGMP-dependent protein kinase (PKG)-related pathways. We hypothesized that SIC would be impaired in MI but restored by activation of PKG, thereby enabling a better response to volume loading in MI. Methods: We conducted experiments in ex vivo rabbit right ventricular papillary muscles under ischemic and non-ischemic conditions as well as pressure-volume hemodynamic evaluations in experimental in vivo MI induced by left anterior descending artery ligation in rats. Results: Acutely stretching muscles ex vivo yielded increased compliance over the next 15 min, but not under ischemic conditions. PKG agonists, but not PKC agonists, were able to partially restore SIC in ischemic muscles. A similar effect was observed with phosphodiesterase-5 inhibitor (PDE5i) sildenafil, which was amplified by joint B-type natriuretic peptide or nitric oxide donor administration. In vivo translation revealed that volume loading after MI only increased cardiac output in rats infused with PDE5i. Contrarily to vehicle, sildenafil-treated rats showed a clear increase in myocardial compliance upon volume loading. Discussion: Our results suggest that ischemia impairs the adaptive myocardial response to acute stretching and that this may be partly prevented by pharmacological manipulation of the cGMP/PKG pathway, namely, with PDE5i. Further studies are warranted to further elucidate the potential of this intervention in the clinical setting of acute myocardial ischemia.

Aim: To evaluate the influence of swimming training on calcium responsiveness of the myocardium of rats with different infarction sizes (MI). Method: female Wistar rats, sedentary sham (SS = 14), sedentary moderate MI (SMI = 8) and sedentary large MI (SLI = 10) were compared to trained sham (TS = 16), trained moderate MI (TMI = 9) and trained large MI (TLI = 10). After 4 weeks of MI, the animals swam for 60 min/day, 5 days/week, for additional 8 weeks. Papillary muscles of the left ventricle were subjected to different concentrations of extracellular calcium. Inotropism was evaluated through the developed tension (DT), the maximum positive value of the first temporal derivation (+Td/td) and the time to peak tension (TPT). Lusitropism was evaluated by the maximum negative value of the first temporal derivation (-Td/td) and time to 50% relaxation (50%TR). Statistical significance was determined using multivariate analysis of variance and a Hotelling T2 test for the absolute power values of all four extracellular calcium concentrations (p < 0.05). Results: MI depressed inotropism (from 17% to 51%) and lusitropism (from 22% to 54%) of the sedentary rats, but exercise attenuated the losses, especially regarding + dT/dt, TPT, -dT/dt and 50%TR. Exercise attenuated the decrease in myocardial responsiveness, proportionally to the size of the MI. Conclusion: Myocardial calcium responsiveness is favorably affected in animals with moderate and large MI after swimming exercise.

The aim of the present study is to characterize the hemodynamics of left ventricular (LV) geometries to examine the impact of trabeculae and papillary muscles (PMs) on blood flow using high performance computing (HPC). Five pairs of detailed and smoothed LV endocardium models were reconstructed from high-resolution magnetic resonance images (MRI) of ex-vivo human hearts. The detailed model of one LV pair is characterized only by the PMs and few big trabeculae, to represent state of art level of endocardial detail. The other four detailed models obtained include instead endocardial structures measuring ≥1 mm2 in cross-sectional area. The geometrical characterizations were done using computational fluid dynamics (CFD) simulations with rigid walls and both constant and transient flow inputs on the detailed and smoothed models for comparison. These simulations do not represent a clinical or physiological scenario, but a characterization of the interaction of endocardial structures with blood flow. Steady flow simulations were employed to quantify the pressure drop between the inlet and the outlet of the LVs and the wall shear stress (WSS). Coherent structures were analyzed using the Q-criterion for both constant and transient flow inputs. Our results show that trabeculae and PMs increase the intra-ventricular pressure drop, reduce the WSS and disrupt the dominant single vortex, usually present in the smoothed-endocardium models, generating secondary small vortices. Given that obtaining high resolution anatomical detail is challenging in-vivo, we propose that the effect of trabeculations can be incorporated into smoothed ventricular geometries by adding a porous layer along the LV endocardial wall. Results show that a porous layer of a thickness of 1.2·10-2 m with a porosity of 20 kg/m2 on the smoothed-endocardium ventricle models approximates the pressure drops, vorticities and WSS observed in the detailed models.

Myocardial relaxation in late systole is enhanced by increasing velocities of lengthening. Given that inorganic phosphate (Pi) can rebind to the force-producing myosin enzyme prior to MgADP release and hasten crossbridge detachment, we hypothesized that myocardial relaxation in late systole would be further enhanced by lengthening in the presence of Pi. Wistar rat left ventricular papillary muscles were attached to platinum clips, placed between a force transducer and a length motor at room temperature, and bathed in Krebs solution with 1.8 mM Ca2+ and varying Pi of 0, 1, 2, and 5 mM. Tension transients were elicited by electrical stimulation at 1 Hz. Peak tension was significantly enhanced by Pi: 0.593 ± 0.088 mN mm-2 at 0 mM Pi and 0.817 ± 0.159 mN mm-2 at 5 mM Pi (mean ± SEM, p < 0.01 by ANCOVA). All temporal characteristics of the force transient were significantly shortened with increasing Pi, e.g., time-to-50% recovery was shortened from 305 ± 14 ms at 0 mM Pi to 256 ± 10 ms at 5 mM Pi (p < 0.01). A 1% lengthening stretch with varying duration of 10-200 ms was applied at time-to-50% recovery during the descending phase of the force transient. Matching lengthening stretches were also applied when the muscle was not stimulated, thus providing a control for the passive viscoelastic response. After subtracting the passive from the active force response, the resulting myofilament response demonstrated features of faster myofilament relaxation in response to the stretch. For example, time-to-70% relaxation with 100 ms lengthening duration was shortened by 8.8 ± 6.8 ms at 0 Pi, 19.6 ± 4.8* ms at 1 mM Pi, 31.0 ± 5.6* ms at 2 Pi, and 25.6 ± 5.3* ms at 5 mM Pi (*p < 0.01 compared to no change). Using skinned myocardium, half maximally calcium-activated myofilaments underwent a 1% quick stretch, and the tension response was subjected to analysis for sensitivity of myosin detachment rate to stretch, g 1, at various Pi concentrations. The parameter g 1 was enhanced from 15.39 ± 0.35 at 0 Pi to 22.74 ± 1.31 s-1/nm at 8 Pi (p < 0.01). Our findings suggest that increasing Pi at the myofilaments enhances lengthening-induced relaxation by elevating the sensitivity of myosin crossbridge detachment due to lengthening and thus speed the transition from late-systole to early-diastole.