Cardiovascular diseases are often associated with impairment in mitochondrial function. However, existing respirometry measuring mitochondrial function are limited by the necessity of fresh tissue samples. This study develops a method with tailored substrate-inhibitor titration (TSIT) of mitochondrial electron transport complexes (ETC) to measure mitochondrial function in frozen cardiac samples using high-resolution respirometry. Briefly, acetyl-CoA is added to fuel the tricarboxylic acid (TCA) cycle for NADH production, enabling complex I (CI)-linked respiratory assessment. NADH is then added to measure maximum CI-linked respiratory capacity, followed by rotenone and succinate to assess complex II (CII)-linked respiratory capacity. TSIT detects mitochondrial functional differences between frozen atrial and ventricular tissue, with comparable results as measured in fresh samples. It also detects cardiac mitochondrial dysfunction across various (patho)physiological mouse models and in human frozen cardiac samples, highlighting its clinical potential. Furthermore, we provides the first evidence for SC formation between the ETC-SCs and the TCA cycle metabolon using blue native electrophoresis, underpinning why TSIT is feasible in frozen tissue. In conclusion, we establish a novel, robust, sensitive and translational method (TSIT) for assessing mitochondrial (dys)function in frozen cardiac samples from various species, enabling flexible analysis of mitochondrial function in both laboratory and clinical settings.

Atrial fibrillation (AF) is a growing cardiovascular epidemic lacking effective treatment. Reactive oxygen species are believed to contribute to AF pathophysiology, yet general antioxidants have limited effectiveness. Since mitochondria are abundant in the heart and a major ROS producer, mitochondrial oxidative stress (MitoOxS) could be a therapeutic target. To determine this, rat inducible immortalized atrial myocytes (iAMs) were tachypaced to mimic AF, followed by assessment of calcium transients, contractility, mitochondrial respiration and morphology, and ROS damage. Cells were pretreated with MitoTEMPO or Sul-238 to assess their protective effects. In Drosophila, heart wall contractions were analyzed to assess arrhythmogenesis after mitochondrial antioxidant pretreatment. Using the GAL4-UAS system, mitochondrial ROS levels and the effect of SOD1 or SOD2 knockdown or overexpression on arrhythmogenesis were evaluated. Tachypacing induced contractile dysfunction and arrhythmogenesis, mitochondrial impairment, and ROS damage in iAMs and increased mitochondrial ROS and arrhythmogenesis in Drosophila. Both MitoTEMPO and Sul-238 treatments prevented mitochondrial dysfunction and arrhythmogenesis in iAMs and rescued arrhythmia in Drosophila. Underscoring the potential to target MitoOxS specifically, SOD2 knockdown promoted arrhythmogenesis in iAMs and Drosophila, whereas SOD2 overexpression rescued tachypacing-induced arrhythmia. MitoOxS is thus a key driver of tachypacing-induced contractile dysfunction and arrhythmia. Mitochondria-targeted antioxidants, such as MitoTEMPO or Sul-238, represent promising therapeutic strategies for AF.

Nicotinamide adenine dinucleotide and β-hydroxybutyrate restore mitochondrial function and metabolic balance, in part by preserving PINK1-Parkin-MFN2-mediated mitophagy. Silencing key mitophagy regulators abolishes these protective effects, identifying mitophagy as a central mechanism linking metabolic interventions to functional recovery and as a potential therapeutic target in atrial fibrillation-associated remodeling.

Cardiovascular diseases are often associated with impairment in mitochondrial function detected by reduced mitochondrial oxygen consumption using high-resolution respirometry. However, existing respirometry protocols are limited by the necessity for fresh tissue samples. This study developed a method with tailored substrate-inhibitor titration (TSIT) of mitochondrial electron transport complexes (ETC) to measure mitochondrial function in frozen cardiac samples using high-resolution respirometry. Briefly, acetyl-CoA was added to fuel the tricarboxylic acid (TCA) cycle for NADH production, enabling complex I (CI)-linked respiratory assessment. NADH was then added to measure maximum CI-linked respiratory capacity, followed by rotenone and succinate to assess complex II (CII)-linked respiratory capacity. TSIT detected mitochondrial functional differences between frozen atrial and ventricular tissue, with comparable results as measured in fresh samples. It also detected cardiac mitochondrial dysfunction across various (patho)physiological mouse models (including aging, ischemia reperfusion, obesity, and CI deficiency) as well as in frozen human donor samples, highlighting its clinical potential. Furthermore, we showed the first evidence for supercomplexes (SCs) formation between ETC-SCs and the TCA cycle metabolon, underpinning TSIT feasibility. In conclusion, we established a novel, robust, sensitive and translational method (TSIT) for assessing mitochondrial (dys)function in frozen cardiac samples from various species, enabling flexible analysis of mitochondrial function in both laboratory and clinical settings.

Background: Cardiac arrhythmia and dysfunction increase with age and are closely associated with declining nicotinamide adenine dinucleotide levels. Nicotinamide, a critical nicotinamide adenine dinucleotide precursor, has shown protective effects in experimental models of aging‐related cardiac diseases. However, its dose‐dependent cardiac‐specific adverse effects and underlying mechanisms warrant investigation. Methods: The effects of nicotinamide (10–100 mmol/L) on the contractility and calcium transient of HL‐1 cardiomyocytes, lifespan, and cardiac function of Drosophila , as well as arrhythmia susceptibility of ex vivo Langendorff mouse hearts were assessed. SERCA2a (sarcoplasmic/endoplasmic reticulum Ca ²⁺ ‐ATPase 2a) acetylation was analyzed by immunoprecipitation followed by Western blotting. Results: Acute perfusion with 10 mmol/L nicotinamide had limited influence on aging‐related atrial fibrillation susceptibility in ex vivo mouse and Drosophila hearts. Short‐term dietary nicotinamide (10 mmol/L) in late life protected against aging‐induced arrhythmia and contractile dysfunction exclusively in male Drosophila . In contrast, lifelong exposure or doses >20 mmol/L caused dose‐dependent adverse cardiac effects, including impaired contractility and calcium transient amplitude and kinetics in HL‐1 cardiomyocyte and shortened lifespan in Drosophila , with impaired excitability and increased arrhythmia in both models. In ex vivo mouse hearts, 100 mmol/L nicotinamide induced ventricular arrhythmia and cardiac arrest. Conclusions: Nicotinamide exhibits dual effects on aging‐related arrhythmia and cardiac dysfunction, with its efficacy highly dependent on dose, duration, and biological context. Although a moderate dose in late life may be protective, chronic or excessive intake induces arrhythmia and impairs cardiac function across species, likely through disrupted cardiac excitability and dysregulated calcium handling. These findings underscore the importance of cautious and context‐specific application of nicotinamide in clinical settings.