Disruption of mitochondria-sarcoplasmic reticulum microdomain connectomics contributes to sinus node dysfunction in heart failure.

The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca2+ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria-SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria-SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload-induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9-mediated knockdown of mitofusin-2 (Mfn2), the mitochondria-SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria-SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria-SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria-SR microdomains resulted in abnormal mitochondrial Ca2+ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria-SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

Protocol to record and quantify the intracellular pH in contracting cardiomyocytes.

Intracellular pH (pHi) plays critical roles in the regulation of cardiac function. Methods and techniques for cardiac pHi measurement have continued to evolve since early 1960s. Fluorescent microscopy is the most recently developed technique with several advantages over other techniques including higher spatial and temporal resolutions, and feasibility for contracting cell measurement. Here, we describe detailed methods for mouse cardiomyocyte isolation, and simultaneous measurement and quantification of pHi and sarcomere length in contracting cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Lyu et al. (2022).

Beat-to-beat dynamic regulation of intracellular pH in cardiomyocytes.

The mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered to maintain a stable intracellular pH (pHi) for normal cardiac function. Even though spatial pHi non-uniformity in cardiomyocytes has been documented, it remains unknown how pHi is regulated to match the dynamic cardiac contractions. Here, we demonstrated beat-to-beat intracellular acidification, termed pHi transients, in synchrony with cardiomyocyte contractions. The pHi transients are regulated by pacing rate, Cl-/HCO3 - transporters, pHi buffering capacity, and ß-adrenergic signaling. Mitochondrial electron-transport chain inhibition attenuates the pHi transients, implicating mitochondrial activity in sculpting the pHi regulation. The pHi transients provide dynamic alterations of H+ transport required for ATP synthesis, and a decrease in pHi 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 pHi handling in excitable cells.

Remodeling of t-system and proteins underlying excitation-contraction coupling in aging versus failing human heart.

It is well established that the aging heart progressively remodels towards a senescent phenotype, but alterations of cellular microstructure and their differences to chronic heart failure (HF) associated remodeling remain ill-defined. Here, we show that the transverse tubular system (t-system) and proteins underlying excitation-contraction coupling in cardiomyocytes are characteristically remodeled with age. We shed light on mechanisms of this remodeling and identified similarities and differences to chronic HF. Using left ventricular myocardium from donors and HF patients with ages between 19 and 75 years, we established a library of 3D reconstructions of the t-system as well as ryanodine receptor (RyR) and junctophilin 2 (JPH2) clusters. Aging was characterized by t-system alterations and sarcolemmal dissociation of RyR clusters. This remodeling was less pronounced than in HF and accompanied by major alterations of JPH2 arrangement. Our study indicates that targeting sarcolemmal association of JPH2 might ameliorate age-associated deficiencies of heart function.