An Optimized Langendorff-free Method for Isolation and Characterization of Primary Adult Cardiomyocytes.

Isolation of adult mouse cardiomyocytes is an essential technique for advancing our understanding of cardiac physiology and pathology, and for developing therapeutic strategies to improve cardiac health. Traditionally, cardiomyocytes are isolated from adult mouse hearts using the Langendorff perfusion method in which the heart is excised, cannulated, and retrogradely perfused through the aorta. While this method is highly effective for isolating cardiomyocytes, it requires specialized equipment and technical expertise. To address the challenges of the Langendorff perfusion method, researchers have developed a Langendorff-free technique for isolating cardiomyocytes. This Langendorff-free technique involves anterograde perfusion through the coronary vasculature by clamping the aorta and intraventricular injection. This method simplifies the experimental setup by eliminating the need for specialized equipment and cannulation of the heart. Here, we introduce an updated Langendorff-free method for isolating adult mice cardiomyocytes that builds on the Langendorff-free protocols developed previously. In this method, the aorta is clamped in situ, and the heart is perfused using a peristaltic pump, water bath, and an injection needle. This simplicity makes cardiomyocyte isolation more accessible for researchers who are new to cardiomyocyte isolation or are working with limited resources. In this report, we provide a step-by-step description of our optimized protocol. In addition, we present example studies of analyzing mitochondrial structural and functional characteristics in isolated cardiomyocytes treated with and without the acute inflammatory stimuli lipopolysaccharide (LPS).

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

FXYD proteins and sodium pump regulatory mechanisms.

The sodium/potassium-ATPase (NKA) is the enzyme that establishes gradients of sodium and potassium across the plasma membrane. NKA activity is tightly regulated for different physiological contexts through interactions with single-span transmembrane peptides, the FXYD proteins. This diverse family of regulators has in common a domain containing a Phe-X-Tyr-Asp (FXYD) motif, two conserved glycines, and one serine residue. In humans, there are seven tissue-specific FXYD proteins that differentially modulate NKA kinetics as appropriate for each system, providing dynamic responsiveness to changing physiological conditions. Our understanding of how FXYD proteins contribute to homeostasis has benefitted from recent advances described in this review: biochemical and biophysical studies have provided insight into regulatory mechanisms, genetic models have uncovered remarkable complexity of FXYD function in integrated physiological systems, new posttranslational modifications have been identified, high-resolution structural studies have revealed new details of the regulatory interaction with NKA, and new clinical correlations have been uncovered. In this review, we address the structural determinants of diverse FXYD functions and the special roles of FXYDs in various physiological systems. We also discuss the possible roles of FXYDs in protein trafficking and regulation of non-NKA targets.

Increased mortality risk associated with serum sodium variations and borderline hypo- and hypernatremia in hospitalized adults.

BACKGROUND: This study aimed to evaluate short-term and long-term mortalities in a cohort of unselected hospitalized patients with serum sodium concentration ([Na+]) variations within and outside of reference range. METHODS: All adult patients admitted to the Mayo Clinic, Rochester, MN, USA from January 2011 to December 2013 (n = 147358) were retrospectively screened. Unique patients admitted during the study period were examined. The main exposure was serum [Na+] variation. Outcome measures were hospital and 1-year all-cause mortalities. RESULTS: A total of 60944 patients, mean age 63 ± 17 years, were studied. On admission, 17% (n = 10066) and 1.4% (n = 852) had hypo- and hypernatremia, respectively. During the hospital stay, 11044 and 4128 developed hypo- and hypernatremia, respectively, accounting for 52.3 and 82.9% of the total hypo- and hypernatremic patients. Serum [Na+] variations of ≥6 mEq/L occurred in 40.6% (n = 24 740) of the 60 944 patients and were significantly associated with hospital and 1-year mortalities after adjusting potential confounders (including demographics, comorbidities, estimated glomerular filtration rate, admission serum [Na+], number of [Na+] measurements and length of hospital stay). Adjusted odds ratios for hospital and 1-year mortalities increased with increasing [Na+] variations in a dose-dependent manner, from 1.47 to 5.48 (all 95% confidence intervals >1.0). Moreover, in fully adjusted models, [Na+] variations (≥6 mEq/L) within the reference range (135-145 mEq/L) or borderline hypo- or hypernatremia (133-137 and 143-147 mEq/L, respectively) compared with 138-142 mEq/L were associated with increased hospital and 1-year mortalities. CONCLUSION: In hospitalized adults, [Na+] fluctuation (≥6 mEq/L) irrespective of admission [Na+] and borderline hypo- or hypernatremia are independent predictors of progressively increasing short- and long-term mortality burdens.

0.9% saline is neither normal nor physiological.

The purpose of this review is to objectively evaluate the biochemical and pathophysiological properties of 0.9% saline (henceforth: saline) and to discuss the impact of saline infusion, specifically on systemic acid-base balance and renal hemodynamics. Studies have shown that electrolyte balance, including effects of saline infusion on serum electrolytes, is often poorly understood among practicing physicians and inappropriate saline prescribing can cause increased morbidity and mortality. Large-volume (>2 L) saline infusion in healthy adults induces hyperchloremia which is associated with metabolic acidosis, hyperkalemia, and negative protein balance. Saline overload (80 ml/kg) in rodents can cause intestinal edema and contractile dysfunction associated with activation of sodium-proton exchanger (NHE) and decrease in myosin light chain phosphorylation. Saline infusion can also adversely affect renal hemodynamics. Microperfusion experiments and real-time imaging studies have demonstrated a reduction in renal perfusion and an expansion in kidney volume, compromising O2 delivery to the renal parenchyma following saline infusion. Clinically, saline infusion for patients post abdominal and cardiovascular surgery is associated with a greater number of adverse effects including more frequent blood product transfusion and bicarbonate therapy, reduced gastric blood flow, delayed recovery of gut function, impaired cardiac contractility in response to inotropes, prolonged hospital stay, and possibly increased mortality. In critically ill patients, saline infusion, compared to balanced fluid infusions, increases the occurrence of acute kidney injury. In summary, saline is a highly acidic fluid. With the exception of saline infusion for patients with hypochloremic metabolic alkalosis and volume depletion due to vomiting or upper gastrointestinal suction, indiscriminate use, especially for acutely ill patients, may cause unnecessary complications and should be avoided. More education regarding saline-related effects and adequate electrolyte management is needed.

Acute Kidney Injury: Tubular Markers and Risk for Chronic Kidney Disease and End-Stage Kidney Failure.

Acute kidney injury (AKI) is a common clinical syndrome directly related to patient short-term and long-term morbidity and mortality. Over the last decade, the occurrence rate of AKI has been increasing, and there has also been a growing epidemic of chronic kidney diseases (CKD) and end-stage kidney disease (ESRD) linked to severe and repeated episodes of AKIs. The detection and management of AKI are currently far from satisfactory. A large proportion of AKI patients, especially those with preexisting CKD, are at an increased risk of non-resolving AKI and progressing to CKD and ESRD. Proposed pathological processes that contribute to the transition of AKI to CKD and ESRD include severity and frequency of kidney injury, alterations of tubular cell phenotype with cells predominantly in the G2/M phase, interstitial fibrosis and microvascular rarification related to loss of endothelial-pericyte interactions and pericyte dedifferentiation. Innate immune responses, especially dendritic cell responses related to inadequate adenosine receptor (2a)-mediated signals, autophagic insufficiency and renin-angiotensin system activation have also been implicated in the progression of AKI and transitions from AKI to CKD and ESRD. Although promising advances have been made in understanding the pathophysiology of AKI and AKI consequences, much more work needs to be done in developing biomarkers for detecting early kidney injury, prognosticating kidney disease progression and developing strategies to effectively treat AKI and to minimize AKI progression to CKD and ESRD.