Inhibition of TGFß1 accelerates regeneration of fibrotic rat liver elicited by a novel two-staged hepatectomy.

Aims: Emerging evidence is demonstrating that rapid regeneration of remnant liver elicited by associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) may be attenuated in fibrotic livers. However, the molecular mechanisms responsible for this process are largely unknown. It is widely acknowledged that the TGFß1 signaling axis plays a major role in liver fibrosis. Therefore, the aims of this study were to elucidate the underlying mechanism of liver regeneration during ALPPS with or without fibrosis, specifically focusing on TGFß1 signaling. Approach: ALPPS was performed in rat models with N-diethylnitrosamine-induced liver fibrosis and no fibrosis. Functional liver remnant regeneration and expression of TGFß1 were analyzed during the ALPPS procedures. Adeno-associated virus-shTGFß1 and the small molecule inhibitor LY2157299 (galunisertib) were used separately or in combination to inhibit TGFß1 signaling in fibrotic rats. Results: Liver regeneration following ALPPS was lower in fibrotic rats than non-fibrotic rats. TGFß1 was a key mediator of postoperative regeneration in fibrotic liver. Interestingly, AAV-shTGFß1 accelerated the regeneration of fibrotic functional liver remnant and improved fibrosis, while LY2157299 only enhanced liver regeneration. Moreover, combination treatment elicited a stronger effect. Conclusions: Inhibition of TGFß1 accelerated regeneration of fibrotic liver, ameliorated liver fibrosis, and improved liver function following ALPPS. Therefore, TGFß1 is a promising therapeutic target in ALPPS to improve fibrotic liver reserve function and prognosis.

Optical Imaging of Isolated Murine Ventricular Myocytes.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

The effects of the ketone body ß-hydroxybutyrate on isolated rat ventricular myocyte excitation-contraction coupling.

ß-hydroxybutyrate is the primary ketone body produced by the body during ketosis and is used to meet its metabolic demands. The healthy adult heart derives most of its energy from fatty acid oxidation. However, in certain diseases, the heart alters its substrate preference and increases its ketone body metabolism. Little is known about the effects of ßOHB on ventricular myocyte excitation-contraction coupling. Therefore, we examined the effects of ketone body metabolism on single cell excitation-contraction coupling during normoxic and hypoxic conditions. Myocytes were isolated from adult rats, cultured for 18 h in RPMI 1640, RPMI 1640 no glucose, and M199, HEPES with/without various amount of ßOHB added. To simulate hypoxia, myocytes were incubated at 1%O2, 5% CO2 for 1 h followed by incubation at atmospheric oxygen (21%O2,5% CO2) for 30 min before recordings. Recordings were obtained using an IonOptix system at 36±1ᵒ C. Myocytes were paced at 0.5, 1, 2, 3, and 4 Hz. We found that exposure to ßOHB had no effect on excitation-contraction coupling. However, culturing cells with ßOHB results in a significant increase in both contraction and calcium in RPMI 1640 media. Dose response experiments demonstrated 0.5 mM ßOHB is enough to increase myocyte contraction in the absence of glucose. However, ßOHB has no measurable effects on myocytes cultured in a nutrient rich media, M199, HEPES. Therefore, ßOHB improves single cell excitation-contraction coupling, is protective against hypoxia, and may be a beneficial adaptation for the heart during periods of nutrient scarcity and or metabolic dysregulation.

Altered myocyte contractility and calcium homeostasis in alpha-myosin heavy chain point mutations linked to familial dilated cardiomyopathy.

Mutations in the human cardiac motor protein beta-myosin heavy chain (ßMHC) have been long recognized as a cause of familial hypertrophic cardiomyopathy. Recently, mutations (P830L and A1004S) in the less abundant but faster isoform alpha-myosin heavy chain (αMHC) have been linked to dilated cardiomyopathy (DCM). In this study, we sought to determine the cellular contractile phenotype associated with these point mutations. Ventricular myocytes were isolated from 2 month male Sprague Dawley rats. Cells were cultured in M199 media and infected with recombinant adenovirus containing the P830L or the A1004S mutant human αMHC at a MOI of 500 for 18 h. Uninfected cells (UI), human ßMHC (MOI 500, 18 h), and human αMHC (MOI 500, 18 h) were used as controls. Cells were loaded with fura-2 (1 µM, 15 min) after 48 h. Sarcomere shortening and calcium transients were recorded in CO2 buffered M199 media (36°±1 C) with and without 10 nM isoproterenol (Iso). The A1004S mutation resulted in decreased peak sarcomere shortening while P830L demonstrated near normal shortening kinetics at baseline. In the presence of Iso, the A1004S sarcomere shortening was identical to the ßMHC shortening while the P830L was identical to the αMHC control. All experimental groups had identical calcium transients. Despite a shared association with DCM, the P830L and A1004S αMHC mutations alter myocyte contractility in completely different ways while at the same preserving peak intracellular calcium.

Left atrial coronary perfusion territories in isolated sheep hearts: implications for atrial fibrillation maintenance.

BACKGROUND: The role of coronary perfusion in the maintenance of atrial fibrillation (AF) electrical sources that anchor to the posterior (wall of the) left atrium (PLA) has been incompletely investigated. We hypothesized that the PLA-pulmonary vein region is perfused by branches originating from both the right and left coronary arteries. OBJECTIVE: The purpose of this study was to evaluate whether branches originating from the right and left coronary arteries could serve as conduits to chemically ablate restricted PLA regions. METHODS: In Langendorff-perfused sheep hearts, the right anterior and left anterior atrial arteries (RAAA and LAAA) and the branches of the left circumflex artery (LCX) were identified as main coronary artery branches perfusing the atria. During sustained AF, 20-mL boluses of cold Tyrode's solution (4°C) was injected into each artery to determine changes in dominant frequency. The injection that yielded the largest dominant frequency decrease indicated the coronary branch to be subsequently perfused with ethanol. Ethanol was selectively injected into the LAAA (n = 4), LCX (n = 4), or RAAA (n = 1). RESULTS: Six of nine AF cases rapidly terminated upon ethanol perfusion. In those hearts and in eight additional preparations (n = 17), Congo red and Evans blue was subsequently perfused into the remaining atrial branches. The perfusion territories were classified as triple-vessel PLA perfusion (n = 4), LAAA-dominant PLA perfusion (n = 5), balanced double-vessel PLA perfusion (n = 5), and LCX or RAAA dominant (n = 3). CONCLUSION: PLA coronary perfusion relies on a variable contribution of right and left coronary branches. Regional irrigation of ethanol in well-delineated PLA perfusion territories enabled ablation of high-frequency sites during AF.