Significance: The heart autoregulates its contractile strength to maintain cardiac output when pumping blood against the mechanical afterload from vascular resistance. Increased afterload in cardiovascular diseases is associated with cardiac arrhythmias. However, the underlying mechanisms remain elusive. Here, we studied the afterload effect on electrophysiology by embedding single cardiomyocytes in a three-dimensional viscoelastic hydrogel to apply afterload during cell contraction. We found that afterload is acutely transduced via nitric oxide synthase 1 (NOS1) signaling to modulate multiple ion channels to prolong action potential, increase Ca ²⁺ transient, and enhance contractility. However, higher afterload causes a discordant alternans that is arrhythmogenic. Hence, our findings reveal an afterload-induced mechano-chemo-electro-transduction pathway that closes feedback loops in cardiac excitation–contraction coupling to enable autoregulation of contractility.

The mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered to maintain a stable intracellular pH (pH_i) for normal cardiac function. Even though spatial pH_i non-uniformity in cardiomyocytes has been documented, it remains unknown how pH_i is regulated to match the dynamic cardiac contractions. Here, we demonstrated beat-to-beat intracellular acidification, termed pH_i transients, in synchrony with cardiomyocyte contractions. The pH_i transients are regulated by pacing rate, Cl⁻/HCO₃^- transporters, pH_i buffering capacity, and β-adrenergic signaling. Mitochondrial electron-transport chain inhibition attenuates the pH_i transients, implicating mitochondrial activity in sculpting the pH_i regulation. The pH_i transients provide dynamic alterations of H⁺ transport required for ATP synthesis, and a decrease in pH_i may serve as a negative feedback to cardiac contractions. Current findings dovetail with the prevailing three known dynamic systems, namely electrical, Ca²⁺, and mechanical systems, and may reveal broader features of pH_i handling in excitable cells.

Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca²⁺ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation–contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods.

Small-conductance Ca ²⁺ -activated K ⁺ channels (SK, K _Ca 2) are gated solely by intracellular microdomain Ca ²⁺ . The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca ²⁺ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca ²⁺ -CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

The heart has two intrinsic mechanisms to enhance contractile strength that compensate for increased mechanical load to help maintain cardiac output. When vascular resistance increases the ventricular chamber initially expands causing an immediate length-dependent increase of contraction force via the Frank-Starling mechanism. Additionally, the stress-dependent Anrep effect slowly increases contraction force that results in the recovery of the chamber volume towards its initial state. The Anrep effect poses a paradox: how can the cardiomyocyte maintain higher contractility even after the cell length has recovered its initial length? Here we propose a surface mechanosensor model that enables the cardiomyocyte to sense different mechanical stresses at the same mechanical strain. The cell-surface mechanosensor is coupled to a mechano-chemo-transduction feedback mechanism involving three elements: surface mechanosensor strain, intracellular ${\mathrm{Ca}}^{2+}$ transient, and cell strain. We show that in this simple yet general system, contractility autoregulation naturally emerges, enabling the cardiomyocyte to maintain contraction amplitude despite changes in a range of afterloads. These nontrivial model predictions have been experimentally confirmed. Hence, this model provides a new conceptual framework for understanding the contractility autoregulation in cardiomyocytes, which contributes to the heart’s intrinsic adaptivity to mechanical load changes in health and diseases.