Mechanical Load Regulates Excitation-Ca²⁺ Signaling-Contraction in Cardiomyocyte

The heart is a smart pump, automatically adjusting its contractile strength in response to mechanical loads placed upon it, via intrinsic adaptations discovered over a century ago.^1,2 When the cardiomyocyte encounters an increase in preload (larger end-diastolic volume) the Frank-Starling effect enhances contractile force and stroke volume, mainly by instantaneous sarcomere-length and myofilament-based effects, independent of Ca²⁺ transient changes. In contrast, when the heart pumps blood against an increase in afterload (larger resistance), the Anrep effect develops over minutes to increase Ca²⁺ transients and enhance contractility. Most prior studies have used stretching methods to control preload on cardiomyocytes; it has been difficult to independently control afterload and the mechanisms underlying the Anrep effect remain unresolved.² We have developed a new methodology to control afterload at the single-myocyte level using our Cell-in-Gel system⁴, and studies how afterload affects the myocyte excitation-Ca²⁺ signaling-contraction (E-C) coupling (Figure 1A).

Figure 1.

(A) Schematic of cardiomyocyte excitation-contraction coupling with mechano-transduction feedback.

(B) Cardiomyocytes were embedded in the polymer matrix made of PVA and 4B-PEG crosslinker.

(C) Mathematical modeling of the Cell-in-Gel system show that the single cardiomyocyte experiences 3D mechanical stresses during auxotonic contraction in the hydrogel.⁴

(D) Patch-Clamp-in-Gel was performed by embedding the cell-electrode in the gel (Da). APs were recorded first when the cell was contracting load-free (LF, black trace in Db), during the gel-forming protocol by adding 10% crosslinker to polymerize PVA (Dc), and after gel formation (Gel, red trace in Db). Bar charts compare cells under load-free vs. in-gel, showing steady-state APD₉₅ (Dd, p=1.9x10^-10) and AP amplitude (De, p=0.45).

(E) Simultaneous measurements of [Ca²⁺]_i and contraction using the gel-dissolve protocol (Ea). Bar charts compare the cells in load-free vs. in-gel, showing diastolic [Ca²⁺]_i (Eb, p=0.17), systolic peak [Ca²⁺]_i (Ec, p=1.2x10^-12), contraction amplitude (Ed, p=3.7x10^-3) and relaxation time (Ee, p=2.12x10^-4).

(F) Cells were embedded in the gels of different stiffness by mixing 10% PVA with crosslinker of indicated concentrations (CL%). Upper panel shows [Ca²⁺]_i transient peak, and lower panel shows contraction amplitude. One-way ANOVA p<0.0001, and Tukey test for pair-wise comparison of neighboring groups.

Statistical tests: The bars show Mean and SEM of each group with indicated number of cells/animals. All groups passed D’Agostino-Pearson normality test. One-way ANOVA test was used for multiple groups comparison; t-test for two groups comparison. p value: p<0.05*, p<0.01**, p<0.001***, ns = not significant.

We embedded freshly isolated rabbit ventricular myocytes in a 3D viscoelastic hydrogel comprising polyvinyl alcohol (PVA) and 4-boronate-polyethylene glycol (4B-PEG) crosslinker (Figure 1B).³ Since the myocytes are embedded at slack length without preload, the Cell-in-Gel system is well-suited for studying afterload effects. Mechanical analyses show that the myocyte contracting in-gel experiences 3D mechanical stresses including longitudinal tension due to cell shortening, transverse compression due to cell broadening, and surface traction with normal and shear stress (Figure 1C).⁴

We further developed a Patch-Clamp-in-Gel technique (Figure 1Da) using a gel-forming protocol. First, we establish patch-clamp of the cell under load-free condition with the myocyte bathed in a modified Tyrode solution containing PVA. Next, add 4B-PEG to crosslink PVA, which embeds the cell-electrode assembly in-gel. Finally, repeat the electrophysiology recordings on the same cell now contracting under afterload in-gel. Figure 1Db shows the action potentials (APs) recorded first in load-free and then under afterload. Upon adding 10% 4B-PEG to 10% PVA, polymerization occurred in only minutes. As the hydrogel became stiffer, the increases of afterload during cell contraction caused progressive increases in AP duration (1Dc). After reaching steady-state, afterload significantly prolonged APD₉₅ (1Dd), with unchanged AP amplitude (1De).

We studied afterload effects on Ca²⁺ signaling and cell contraction using a gel-dissolve protocol (Figure 1E). First, we measured cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and sarcomere length shortening simultaneously while the myocyte was embedded in-gel and paced to perform E-C coupling under afterload. Next, sorbitol (1% w/v) was added to the perfusion solution to dissolve the hydrogel. Finally, we repeated experiments on the same cell, now contracting in load-free condition. These self-control experiments show that afterload did not alter diastolic [Ca²⁺]_i (1Eb), but increased systolic Ca²⁺ transients (1Ec), reduced sarcomere shortening magnitude (1Ed), and slowed relaxation (1Ee). Thus, afterload-induced mechano-chemo-transduction (MCT) regulates the Ca²⁺ signaling system, causing MCT-Ca²⁺ gain to enhance contractility.

The afterload effects on electrophysiology and Ca²⁺ signaling provide feedback loops in the dynamic system of E-C coupling, which may enable autoregulation. To test this hypothesis, we systematically tuned afterload levels using hydrogels of different stiffness (mixing 10% PVA with different crosslinker concentrations, CL%). Cardiomyocytes showed progressively larger Ca²⁺ transients under higher afterload in stiffer gels (Figure 1F, top panel; CL5%–10%, gel elastic shear modulus 1–10 kPa). Remarkably, the increases of Ca²⁺ transients enabled cardiomyocytes to maintain relatively stable contraction amplitude despite load increases (bottom panel). It was not until very high afterload (CL11%–15%, gel elastic shear modulus 11–15 Pa) that myocyte contraction declined with reduced MCT-Ca²⁺ gain. In conclusion, our studies reveal mechano-chemo-electro-transduction (MCET) feedbacks in the dynamic system of cardiac excitation-Ca²⁺ signaling-contraction coupling, which enable autoregulation of contractility at the single-myocyte level independent of neurohormonal influences. The Cell-in-Gel methodology provides a powerful tool for further dissection of MCET molecular pathways that underlie the heart’s intrinsic adaptive responses to mechanical loading in health and diseases.

Data Availability.

The methods, data, and materials of this study are available upon request to the corresponding author and through the university material transfer agreement.

New Zealand White rabbits, 4–6 months old male, were purchased from Charles River Laboratories (Wilmington, MA) and used to isolate cardiomyocytes using standard enzymatic technique. All animal procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH) and the protocols approved by the University of California Davis Institutional Animal Care and Use Committee (IACUC).