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.

RNA Profiling in Human and Murine Transplanted Hearts: Identification and Validation of Therapeutic Targets for Acute Cardiac and Renal Allograft Rejection.

Acute cellular rejection (ACR) is the adverse response of the recipient's immune system against the allogeneic graft. Using human surveillance endomyocardial biopsies (EMBs) manifesting ACR and murine allogeneic grafts, we profiled implicated microRNAs (miRs) and mRNAs. MiR profiling showed that miR-21, -142-3p, -142-5p, -146a, -146b, -155, -222, -223, and -494 increased during ACR in humans and mice, whereas miR-149-5p decreased. mRNA profiling revealed 70 common differentially regulated transcripts, all involved in immune signaling and immune-related diseases. Interestingly, 33 of 70 transcripts function downstream of IL-6 and its transcription factor spleen focus forming virus proviral integration oncogene (SPI1), an established target of miR-155, the most upregulated miR in human EMBs manifesting rejection. In a mouse model of cardiac transplantation, miR-155 absence and pharmacological inhibition attenuated ACR, demonstrating the causal involvement and therapeutic potential of miRs. Finally, we corroborated our miR signature in acute cellular renal allograft rejection, suggesting a nonorgan specific signature of acute rejection. We concluded that miR and mRNA profiling in human and murine ACR revealed the shared significant dysregulation of immune genes. Inflammatory miRs, for example miR-155, and transcripts, in particular those related to the IL-6 pathway, are promising therapeutic targets to prevent acute allograft rejection.