Assessing fatty acid oxidation flux in rodent cardiomyocyte models

The healthy adult heart primarily relies on fatty acid oxidation (FAO) for energy production but instantaneously adapts its substrate preference in response to physiological or pathological challenges. Accurate FAO measurements are crucial to investigate early metabolic (mal)adaptations. While measurements in intact cardiomyocytes offer greater physiological relevance, current FAO protocols mainly employ cell-free systems and/or require expensive equipment. Here, we present an easy-to-use, inexpensive, and sensitive method to measure, compare and modulate FAO in various cardiomyocyte models. Basal FAO was 2-fold higher in fresh versus cultured adult rat cardiomyocytes (aRCM), while OXPHOS protein levels were maintained. Basal FAO was higher in cultured (3-fold) and fresh (8-fold) aRCM, versus widely used neonatal rat cardiomyocytes (nRCM) and mouse HL1 cardiomyocytes. Moreover, we utilized chemical and pharmacological treatments in order to modulate the FAO flux at different cellular signalling levels. Our data indicate that caution should be taken when studying metabolism in nRCM and HL1 cell models, as these display significantly lower FAO than aRCM. Accurate FAO measurement in cultured aRCM opens new avenues for studying the complex cardiomyocyte metabolic responses to mechanical, nutritional, pharmacological, and genetic manipulations.


Animals
Male Lewis rats (300-450 g), 12-week-old male Zucker fatty and lean rats (Zuc-Lepr fa fa/fa and ZUC-Lepr fa fa/+, respectively) and 3-day-old Wistar neonatal rats were purchased from Charles River Laboratories (The Netherlands) and used for cardiomyocyte isolation. The animals were housed in a controlled environment (21-22°C) with a 12:12 hour light/dark cycle and free access to food and water.

RNA analysis
Total RNA was isolated from aRCM with the miRVana microRNA isolation kit (Ambion, Austin, TX). A miScript PCR system (Qiagen) was used for cDNA synthesis. SYBR Green® quantitative PCR was performed on a Bio-Rad iCycler (Hercules, CA) to determine gene expression levels of ucp2, acsl1, acox1, cd36 genes with the respective primers: Ucp2 Fw:

2.
In order to be included in the experiment it was required that >80% of the aRCM were rod-shaped and excluded trypan blue.

3.
The corresponding BSA-palmitate ratio is 0.75, thus 2x below the K M for the uptake process 2 . Given that the trans-sarcolemmal uptake is the rate controlling step in the total oxidation process of FA, this indicates that under these conditions, changes in FAO flux can be sensitively detected.

A zero time control (ZTC) condition is a correction for the background signal, and
therefore serves as a negative control for the assay. There should be at least one incubation per each experiment. In the ZTC, the stop solution is added just before addition of DLOx. When performing the calculation of FAO, the counts of this ZTC have to be substracted from the radioactive counts of each incubation.

5.
We have previously shown that 14 CO 2 production from labeled palmitate was detectable after 10 minutes and increased linearly with time for at least 2 hours 2 , indicating that oxidation, as measured during our current 30-min protocol, only takes place during the 10 to 30 min time period.

6.
Calculation: 28 pmol/mg protein/min equals ~5.0 nmol/g wet weight per min (as 1 gram wet weight equals ~178 mg protein 2 ). After correcting for the administered palmitate concentration (e.g. 500 µM is used in O'Donnell's 3 versus 100 µM in our study) and considering that in vivo cells are contracting and thus have higher FAO rates than ex vivo cells (i.e. 4 Hz stimulation of aRCM led to a 2.8 fold upregulation in FAO 4 ), the FAO value in our study compares to ~70 nmol / g wet weight / min. This is ~0.56 µmol/g dry weight per min (as 1 gram dry weight equals 8 gram wet weight 5 ).

7.
Oligomycin is a specific inhibitor of mitochondrial F 1 F 0 -ATP synthase and is often shown to inhibit FAO. However, Ylitalo et al. 6 showed that in isolated rat heart mitochondria there is a range of oligomycin concentrations at which there are elevated intracellular AMP levels, without any inhibitory actions on oxygen consumption rates.
The increased intracellular AMP/ATP ratio then leads to AMPK activation, phosphorylation of ACC and subsequent de-inhibition of CPT-I, thereby leading to increased FAO. Figure S1. Figure S1. Full-length blot of OXPHOS protein that is shown in the main article in figure 1D. Western blot analysis was performed in lysates of neonatal rat cardiomyocytes (nRCM), HL1 cardiomyocytes (HL1), and fresh and cultured adult rat cardiomyocytes (aRCM). The lane where the molecular weight marker was loaded is marked with MW. Rat heart mitochondria served as positive control (P).