Marterstock M, Schauer A, Maslakova A
… +7 more, Opitz A, Dieterich P, Speier S, Kämmerer S, Mirtschink P, Kopaliani I, Deussen A
Acta Physiol (Oxf)
· 2026 Jun · PMID 42136002
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AIM: Echocardiographic assessment of diastolic function in mice remains challenging because parameters translated from clinical practice are constrained by murine physiology. In particular, parameters derived from early...AIM: Echocardiographic assessment of diastolic function in mice remains challenging because parameters translated from clinical practice are constrained by murine physiology. In particular, parameters derived from early (E) and late (A) transmitral filling velocities are frequently compromised by wave fusion at higher heart rates. Here, we quantified these limitations and evaluated an alternative method for measuring isovolumetric relaxation time (IVRT), a marker reflecting ventricular relaxation and a key element of established algorithms specific for murine diastolic function assessment. METHODS: In a non-A4C-view-dependent echocardiographic measurement (NAEM) approach, speckle-tracking analysis of standard brightness-mode (B-mode) cine loops from the parasternal long-axis view (PSLAX) enabled E/A ratio and IVRT assessment. Mice with unimpaired heart function, heart failure with preserved ejection fraction (HFpEF), or heart failure with reduced ejection fraction (HFrEF) (n = 8 each) underwent standard Doppler-based and NAEM-based measurement of E/A ratio and IVRT. RESULTS: In mice with unimpaired heart function and HFpEF, heart rate-dependent E and A wave fusion occurred at an estimated threshold of approximately 460 bpm, whereas advanced HFrEF led to frequent fusion independent of heart rate. NAEM-derived IVRT showed strong agreement with the Doppler reference (r = 0.9339, p < 0.0001), minimal bias (-1.4 ms), and similar inter-observer variability. In contrast, NAEM-derived E/A ratio did not show adequate performance. CONCLUSION: These findings further support the shift toward specific algorithms for diastolic function assessment in murine echocardiography. The validated NAEM approach supports application of such protocols without dedicated Doppler recordings by using PSLAX B-mode cine loops, thereby lowering technical barriers for future studies and enabling robust retrospective analysis of existing datasets.
Gaglianone RB, Lamboley CR, Dick T
… +3 more, Meizoso-Huesca A, Singh DP, Launikonis BS
Acta Physiol (Oxf)
· 2026 Jun · PMID 42135879
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AIM: The dystrophic mdx mouse is a widely used model of Duchenne muscular dystrophy. Altered Ca handling is a key feature, including increased Ca leak through the ryanodine receptor (RyR1's), the primary Ca release chann...AIM: The dystrophic mdx mouse is a widely used model of Duchenne muscular dystrophy. Altered Ca handling is a key feature, including increased Ca leak through the ryanodine receptor (RyR1's), the primary Ca release channel in skeletal muscle. Such leak has important downstream consequences for intracellular Ca homeostasis. Here, we quantified basal compartmentalized Ca levels in mdx muscle compared with wild-type (WT). METHODS: Single extensor digitorum longus muscle fibers from WT and mdx mice were mechanically skinned. Transverse tubule Ca dynamics were assessed using confocal microscopy with fluorescent Ca indicators during caffeine-induced RyR1-mediated Ca release. Sarcoplasmic reticulum (SR) and mitochondrial Ca contents were quantified using established depletion protocols combined with force measurements. RESULTS: Consistent with previous reports, mdx fibers exhibited increased RyR1 Ca leak. Absolute quantification revealed a reduction in SR Ca content accompanied by a ~4-fold increase in mitochondrial Ca content. These shifts indicate a redistribution of intracellular Ca, triggered by the RyR1 Ca leak to lower SR Ca content and increase the Ca permeability of the t-system membrane, leading to an elevation in cytoplasmic and mitochondrial Ca levels in mdx muscle. CONCLUSION: Redistribution of Ca is a regulated process, proportional to RyR1 Ca leak. In mdx muscle fibers, there is reduced SR and elevated mitochondrial and cytoplasmic Ca compared to WT fibers. These alterations contribute to the dystrophic muscle pathology, likely through promotion of oxidative stress through increased reactive oxygen species production.
Cavalieri R, Dela Rosa MAS, Cotrim CA
… +5 more, Copeman D, Aris M, Eke C, Staggs-Sandy H, Crichton PG
Acta Physiol (Oxf)
· 2026 Jun · PMID 42130299
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Uncoupling Protein 1 (UCP1) is a defining feature of brown fat and facilitates the specialized ability of the tissue to generate heat in the process of non-shivering thermogenesis. The protein is activated by fatty acids...Uncoupling Protein 1 (UCP1) is a defining feature of brown fat and facilitates the specialized ability of the tissue to generate heat in the process of non-shivering thermogenesis. The protein is activated by fatty acids, which overcome its inhibition by purine nucleotides, to catalyze proton leak across the mitochondrial inner membrane, uncoupling nutrient oxidation from ATP production to release energy as heat. Thermogenesis through this process contributes to thermoregulation in many mammals and can promote nutrient turnover in humans to support metabolic health. UCP1 is a member of the mitochondrial carrier family of solute exchangers. For many years, its underlying mechanisms of activity and regulation have remained unclear. However, recent cryo-EM structures of UCP1 have clarified details on nucleotide inhibition and, with advances in our understanding of the mitochondrial carrier transport mechanism, provided important molecular constraints to rationalize how the protein may operate. Here, we review the molecular nature of UCP1, re-evaluating past structure-function relations in this structural context. Key carrier features and putative novel bonding that likely support state changes in the protein and proton leak activity are highlighted, as well as new hypotheses to explain subtleties in purine nucleotide binding discrimination.
Aalkjær C, Dam VS, Nilsson H
… +2 more, Boedtkjer DB, Matchkov V
Acta Physiol (Oxf)
· 2026 Jun · PMID 42121280
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AIM: Ca-activated Cl- conductances are present in many cell types and are important for regulating membrane potential as well as other cellular functions. TMEM16A is widely accepted as the principal molecular basis for C...AIM: Ca-activated Cl- conductances are present in many cell types and are important for regulating membrane potential as well as other cellular functions. TMEM16A is widely accepted as the principal molecular basis for Ca-activated Cl- conductances, but also members of the bestrophin family may be important for some Ca-activated Cl- conductances. METHODS: In this review, we discuss the possibility that members of the two families, bestrophins and TMEM16A, may interact. RESULTS: In the cardiovascular system, the evidence is strongest. Here (1) TMEM16A and bestrophin-3 are closely located in the membrane, (2) TMEM16A may regulate the expression of bestrophin-3, and (3) a cGMP dependent Ca-activated Cl- conductance is dependent on both TMEM16A and bestrophin-3. Outside the cardiovascular system, both TMEM16A and bestrophins are implicated in the regulation of cellular Ca independently of membrane potential, and both proteins are importantly associated with inflammation and pain transmission. However, it seems that little testing of a direct interaction of TMEM16A and bestrophins has been published. CONCLUSION: We conclude that there is evidence for direct interaction between TMEM16A and bestrophins in the cardiovascular system, and it will be important to determine whether similar interactions extend to other tissues.
BACKGROUND: Pathological cardiac hypertrophy is a key risk factor for heart failure (HF). Illustrating the pathogenesis of cardiac hypertrophy may contribute to the treatment of HF. Studies have emphasized that protein u...BACKGROUND: Pathological cardiac hypertrophy is a key risk factor for heart failure (HF). Illustrating the pathogenesis of cardiac hypertrophy may contribute to the treatment of HF. Studies have emphasized that protein ubiquitination is a critical event in HF. In this study, we investigated the role of an E3 ubiquitin ligase, RNF123, in HF induced by angiotensin II (Ang II) infusion and transverse aortic constriction (TAC) surgery. METHODS: Heart failure was induced by Ang II infusion or TAC surgery in wild-type and RNF123 knockout mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis combined with co-immunoprecipitation (Co-IP) was used to identify PRDX1 as an interacting protein of RNF123. RESULTS: The expression of RNF123 was significantly increased in cardiomyocytes of mice subjected to Ang II infusion or TAC surgery. RNF123 deficiency mitigated cardiac hypertrophy and dysfunction induced by Ang II infusion or TAC operation in mice. In vitro, RNF123 knockdown attenuated Ang II-induced hypertrophy, whereas RNF123 overexpression exacerbated the pathological alterations in neonatal rat ventricular myocytes (NRVMs). Mechanistically, LC-MS/MS and Co-IP assays revealed that RNF123 directly bound to the N-terminal domain of PRDX1 and added a K48-linked ubiquitin chain at the K7 site of PRDX1, subsequently facilitating PRDX1's proteasomal degradation. RNF123-mediated degradation of PRDX1 increased the ROS level in cardiomyocytes, driving the pathogenesis of myocardial hypertrophy. CONCLUSIONS: Our findings identified that cardiomyocyte RNF123 mediates pathological cardiac hypertrophy via ubiquitinating PRDX1 and highlighted that targeting RNF123 may represent a promising therapeutic strategy for HF.
AIM: Chronic high-fat diet (HFD) consumption combined with lipopolysaccharide (LPS) challenge promotes microglial hyperactivation, brain inflammation, reduced neurogenesis, and depression-like behavior. LPS also increase...AIM: Chronic high-fat diet (HFD) consumption combined with lipopolysaccharide (LPS) challenge promotes microglial hyperactivation, brain inflammation, reduced neurogenesis, and depression-like behavior. LPS also increases adenosine triphosphate (ATP) release from immune and dying cells, activating microglia through the purinergic P2X7R receptor. However, the effects of P2X7 inhibitor on microglial hyperactivation, brain inflammation, neurogenesis, and depression-like behavior in obese models challenged with LPS remain unclear. METHODS: Sixty-four male Wistar rats were fed either normal diet or HFD for 12 weeks and subsequently received an intraperitoneal (IP) injection of normal saline or LPS (500 μg/kg). LPS-treated rats were then given saline, minocycline (45 mg/kg, twice, IP), or the P2X7R inhibitor JNJ-55308942 (30 mg/kg, single dose, orally). Depression- and anxiety-like behaviors were assessed 24 h later. RESULTS: LPS alone induced pronounced peripheral and brain inflammation, elevated circulating LPS, microglial hyperactivation, increased ATP/P2X7-mediated neuroinflammation, excessive C1q-mediated synaptic pruning, and mood-related behavioral deficits. Chronic HFD additionally induced metabolic disturbances, oxidative stress, blood-brain barrier disruption, and reduced neurogenesis. Combined HFD and LPS exposures further amplified brain pathologies and the severity of mood-related deficits. P2X7R inhibitor effectively reduced oxidative stress, suppressed ATP/P2X7-mediated neuroinflammation, limited aberrant synaptic pruning, restored neurogenesis, and improved behaviors. Minocycline improved behavioral outcomes primarily by reducing endotoxemia and inflammation. CONCLUSION: The comparable neuroprotection produced by JNJ-55308942 and minocycline suggests that ATP/P2X7-mediated neuroinflammation plays a major role in regulating brain pathologies in HFD-fed rats, followed by LPS challenge. These findings suggest P2X7 signaling as a promising therapeutic target for depression and inflammation-associated neuropsychiatric disorders.
Acta Physiol (Oxf)
· 2026 Jun · PMID 42080290
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The use of a startling acoustic stimulus during a simple reaction time task results in the rapid initiation of a prepared response at extremely short latencies (< 80 ms). This so-called "StartReact effect" has been incre...The use of a startling acoustic stimulus during a simple reaction time task results in the rapid initiation of a prepared response at extremely short latencies (< 80 ms). This so-called "StartReact effect" has been increasingly employed to probe subcortical contributions to response preparation, as it is thought to occur due to increased activation in reticulospinal pathways associated with engagement of the startle reflex. However, the lack of an agreed-upon definition of what exactly constitutes a StartReact effect, combined with differences in methodological protocols, has resulted in inconsistent interpretation of experimental results. Based on a comprehensive review of the literature, including evidence for the physiological mechanism underlying the effect, we propose that the clearest definition of the StartReact effect is "the early and involuntary triggering of a prepared movement in the presence of a startle reflex". Reflexive startle activity has been shown to be strongly associated with involuntary response initiation and avoids other potential confounding variables that have been shown to speed reaction time. Here we argue that classification of trials based on startle-related activation in sternocleidomastoid is the most robust method to confirm a StartReact effect. Special situations, such as pre-pulse inhibition, movements involving musculature that require additional considerations, and lowered response preparation levels, are also considered with regards to how to confirm the presence of a StartReact effect. Future directions, including the use of a StartReact protocol as a potential adjuvant therapy for movement disorders, are discussed.
Jing-Ran Z, Hui-Xiao F, Xing-Yu L
… +4 more, Hong-Fei Z, Tian-Min D, Bai-Yan L, Di W
Acta Physiol (Oxf)
· 2026 Jun · PMID 42071178
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AIM: To investigate how tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na channels coordinately fine-tune action potential (AP) depolarization and firing capability in rat nodose visceral sensory neuro...AIM: To investigate how tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na channels coordinately fine-tune action potential (AP) depolarization and firing capability in rat nodose visceral sensory neurons. METHODS: APs were recorded by ruptured-patch current clamp in unmyelinated C-type and myelinated Ah-type neurons from isolated and sliced nodose ganglia. Voltage derivatives and displacement current phase plots were used to determine the kick-in voltage of TTX-R following TTX-S activation. Myelinated A-type neurons, which express TTX-S exclusively, served as a model for dynamic current-clamp (DCC) simulation, in which gNa0 (TTX-S) and gNa1 (TTX-R) were injected separately or in combination. RESULTS: Voltage derivatives and phase plots revealed a biphasic upstroke in C- and Ah-type neurons, indicating sequential TTX-S then TTX-R activation. The TTX-R kick-in voltage was more negative in Ah-type than in C-type neurons and was strongly inversely correlated with the maximal upstroke velocity. DCC faithfully reproduced both AP types; TTX-R reactivation generated the C-type repolarization hump, and AP peak was preserved through proportional gNa0/gNa1 compensation. Increasing the integrated step size of gNa1 delayed TTX-R recruitment, reduced the second Na peak, and progressively impaired repetitive firing, whereas the TTX-S peak remained unchanged. CONCLUSION: TTX-S and TTX-R Na channels coordinate AP depolarization sequentially and compensatorily: TTX-S initiates the upstroke, whereas TTX-R is recruited later and reactivates during repolarization to constrain firing frequency. Combining patch-clamp with DCC simulation provides novel insight into visceral sensory neuron excitability.
AIM: The fusion of myoblasts to form multinucleated myofibres is a key step in the regeneration of skeletal muscle following injury. In this study, we elucidate how GRP94 regulates myoblast fusion during skeletal muscle...AIM: The fusion of myoblasts to form multinucleated myofibres is a key step in the regeneration of skeletal muscle following injury. In this study, we elucidate how GRP94 regulates myoblast fusion during skeletal muscle regeneration. METHODS: Skeletal muscle injury and regeneration models were established in mice, and myogenic differentiation was induced in C2C12 myoblasts in vitro. GRP94 function was inhibited pharmacologically or reduced by downregulating its expression. Muscle regeneration, myoblast fusion, and Myomaker expression were assessed by hematoxylin and eosin staining, Western blotting, and immunofluorescence. The interaction between GRP94 and Myomaker and its regulatory mechanisms were analyzed using immunoprecipitation and ubiquitin-proteasome assays. RESULTS: Inhibition of GRP94 delayed muscle regeneration in vivo, resulting in smaller regenerating myofibres and reduced myoblast fusion in vitro. GRP94 suppression decreased Myomaker expression, disrupted its subcellular localisation, and impaired its membrane trafficking. Mechanistically, GRP94 interacted with Myomaker, facilitated its post-translational translocation, and protected it from ubiquitin-mediated degradation. CONCLUSION: GRP94 promotes the post-translational translocation of Myomaker and delays its degradation via the ubiquitin-proteasome pathway. It thereby regulates myoblast fusion and skeletal muscle regeneration, providing new strategies and a basis for the treatment of muscle regeneration disorders and muscle-related diseases.
Flessner R, Ilie A, Han L
… +4 more, Boucher A, McKinney RA, Sharif-Naeini R, Orlowski J
Acta Physiol (Oxf)
· 2026 Jun · PMID 42051037
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AIM: The alkali cation/proton exchanger NHE6/SLC9A6 regulates luminal pH homeostasis and trafficking of recycling endosomes in most tissues, especially neurons. Loss-of-function mutations in NHE6 cause Christianson Syndr...AIM: The alkali cation/proton exchanger NHE6/SLC9A6 regulates luminal pH homeostasis and trafficking of recycling endosomes in most tissues, especially neurons. Loss-of-function mutations in NHE6 cause Christianson Syndrome, an X-linked neurodevelopmental and neurodegenerative disorder; however, the underlying molecular and cellular mechanisms remain unclear. Here, we describe a new role for NHE6 as a scaffolding platform for recruiting and delivering signaling molecules to the plasma membrane. METHODS: The yeast two-hybrid system was used to screen a human brain cDNA library for proteins that bind to the cytoplasmic C-terminus of NHE6. RESULTS: Cyclin-dependent kinase 5 (CDK5) was identified as a putative interacting partner. CDK5 is widely expressed and phosphorylates diverse proteins involved in vital processes, including receptor signaling, cytoskeletal organization, endocytosis, exocytosis, and apoptosis. Formation of a NHE6/CDK5 complex was confirmed by biochemical assays and microscopy using Chinese hamster ovary AP-1 and human neuroblastoma SH-SY5Y cells. CDK5, in a complex with its activator subunit p35/CDK5R1, did not directly phosphorylate or regulate the membrane trafficking of NHE6. By contrast, NHE6 expression enhanced the localization of CDK5 and p35 to endosomal- and plasmalemmal-enriched membrane fractions and elevated cell surface accumulation of the CDK5-regulated transient receptor potential V1 (TRPV1) cation channel. CONCLUSIONS: These data indicate that NHE6, aside from its main pH-regulatory function, can act concomitantly as a scaffold for recruitment of CDK5/p35 to endosomes and the plasma membrane where the kinase is now primed to activate neighboring effectors important for cell function.
Essigke D, Kalo MZ, Wörn M
… +6 more, Li XQ, Bohnert BN, Schork A, Birkenfeld AL, Ott T, Artunc F
Acta Physiol (Oxf)
· 2026 Jun · PMID 42037165
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AIMS: The epithelial sodium channel ENaC consists of the subunits α, β, and γ and is activated at an individual channel level by proteolytic processing. Murine γENaC contains a distal polybasic tract 186 mediating proteo...AIMS: The epithelial sodium channel ENaC consists of the subunits α, β, and γ and is activated at an individual channel level by proteolytic processing. Murine γENaC contains a distal polybasic tract 186 mediating proteolytic ENaC activation by serine proteases in vitro. The relevance of ENaC activation at this cleavage site for sodium homeostasis in vivo is unknown. METHODS: Mice were generated carrying a mutation of the distal polybasic tract (RKRK186QQQQ or γENaC) using CRISP/Cas9. Sodium homeostasis and proteolytic processing of γENaC were investigated under a low sodium diet, pharmacological ENaC blockade, and induction of nephrotic syndrome. RESULTS: Under control conditions, the response to bolus amiloride was reduced in γENaC mice compared to γENaC mice. Under a low sodium diet for 4 days, urinary sodium excretion was similarly lowered in both genotypes; however, γENaC mice required significantly higher plasma aldosterone concentrations. Both genotypes were similarly tolerant to amiloride exposure for 4 days and developed similar sodium retention and body weight gain after induction of nephrotic syndrome. Proteolytic processing of γENaC leading to increased expression of distally cleaved γENaC at ~54 kDa was stimulated in both γENaC and γENaC mice under all interventions without an appreciable difference in the migration pattern. CONCLUSION: Mice harboring the RKRK186QQQQ mutation of the distal polybasic tract develop hyperaldosteronism under a low sodium diet, pointing to the relevance of this tract for sodium preservation. However, proteolytical processing of γENaC in these mice appears to be compensated for by the involvement of other adjacent cleavage sites.
Bozzini N, Cortini M, Righi A
… +7 more, Grigoriadis AE, Dack M, Ilieva E, Torricelli F, Manicardi V, Baldini N, Avnet S
Acta Physiol (Oxf)
· 2026 Jun · PMID 42036820
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AIM: The tumor microenvironment in solid tumors is characterized by extracellular acidosis, which promotes cancer aggressiveness. In osteosarcoma, the most common primary bone cancer, a highly acidic tumor microenvironme...AIM: The tumor microenvironment in solid tumors is characterized by extracellular acidosis, which promotes cancer aggressiveness. In osteosarcoma, the most common primary bone cancer, a highly acidic tumor microenvironment is associated with metastasis and poor prognosis, partly due to metabolic rewiring, including changes in lipid pathways such as those involving sphingosine-1-phosphate, a bioactive sphingolipid. Sphingosine-1-phosphate has been previously implicated in histone deacetylase inhibition and gene activation. Here, we investigated whether acidosis induces nuclear sphingosine-1-phosphate accumulation via sphingosine kinase 2, leading to epigenetic activation of oncogenes like FOS in osteosarcoma. METHODS: Osteosarcoma spheroids were cultured under neutral or acidic conditions. Histone H3 acetylation was assessed by capillary Western blotting. FOS expression and FOS nuclear localization were analyzed. Sphingosine-1-phosphate's role was addressed through sphingosine kinase 2 silencing and inhibition (ABC294640). Functional effects were measured using colony formation assays. Patient-derived OS tissues (n = 7) were analyzed for correlations between acidity markers (LAMP2, V-ATPase), sphingosine kinase 2, and FOS expression. RESULTS: Acidosis increased both sphingosine kinase 2 mRNA expression after 24 h and histone H3 acetylation, which followed progressive FOS upregulation and nuclear FOS accumulation. Sphingosine kinase 2 inhibition or silencing reduced these effects and impaired clonogenicity. In patient tissues, sphingosine kinase 2 levels correlated with acidosis markers and FOS expression. CONCLUSIONS: We identified a novel mechanism where acidosis stimulates both nuclear sphingosine kinase 2 to synthesize sphingosine-1-phosphate and histone H3 acetylation, ultimately leading to FOS transcription. Targeting this axis decreased clonogenesis, underscoring its therapeutic potential in osteosarcoma and potentially other acid-adapted cancers.
Cuesta-Corral M, Montoro-Garrido A, Romero-Miranda A
… +11 more, Islas F, Ramchandani B, Gredilla R, Fernández-Irigoyen J, Santamaría E, Delgado-Valero B, Jiménez-González S, Rodrigues Díez R, Nieto ML, Cachofeiro V, Martínez-Martínez E
Acta Physiol (Oxf)
· 2026 Jun · PMID 42033100
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AIM: Myocardial infarction (MI) is one of the leading causes of death worldwide. MI is associated with cardiac structural and functional alterations. Among these, cardiac fibrosis may be significantly influenced by mitoc...AIM: Myocardial infarction (MI) is one of the leading causes of death worldwide. MI is associated with cardiac structural and functional alterations. Among these, cardiac fibrosis may be significantly influenced by mitochondrial dysfunction. We sought to evaluate whether the injection of functional mitochondria from healthy muscle could improve the detrimental consequences of MI. METHODS: Male Wistar rats were submitted to MI through the ligature of the left anterior descending coronary artery. Animals subjected to a sham operation (the same surgical procedure without fastening of the suture that passes through the LAD) were included as a reference group (Sham). At the time of surgery, either vehicle (PBS) or isolated mitochondria (equivalent to 180 μg of mitochondrial protein in 75 μL of vehicle) were directly injected into the myocardium around the ligation to half of the animals in each group. Animals were sacrificed 4 weeks after both MI induction and the evaluation of cardiac and systolic functions. RESULTS: Cardiac mitochondrial transplantation was able to prevent the decrease in systolic function and the development of cardiac fibrosis in MI rats. These beneficial effects were accompanied by a reduction in cardiac hypertrophy, oxidative stress, endoplasmic reticulum stress activation, and inflammatory markers. We also evaluated the effects of mitochondrial transplantation by a proteomic analysis. In addition, cardiac mitochondrial transplantation was able to prevent the development of renal alterations observed in MI rats. CONCLUSIONS: The data reveal novel mechanisms of mitochondrial transplantation effects and emerge as a novel therapeutic strategy under chronic diseases such as MI.