Tachycardiomyopathy entails a dysfunctional pattern of interrelated mitochondrial functions.

Tachycardiomyopathy is characterised by reversible left ventricular dysfunction, provoked by rapid ventricular rate. While the knowledge of mitochondria advanced in most cardiomyopathies, mitochondrial functions await elucidation in tachycardiomyopathy. Pacemakers were implanted in 61 rabbits. Tachypacing was performed with 330 bpm for 10 days (n = 11, early left ventricular dysfunction) or with up to 380 bpm over 30 days (n = 24, tachycardiomyopathy, TCM). In n = 26, pacemakers remained inactive (SHAM). Left ventricular tissue was subjected to respirometry, metabolomics and acetylomics. Results were assessed for translational relevance using a human-based model: induced pluripotent stem cell derived cardiomyocytes underwent field stimulation for 7 days (TACH-iPSC-CM). TCM animals showed systolic dysfunction compared to SHAM (fractional shortening 37.8 ± 1.0% vs. 21.9 ± 1.2%, SHAM vs. TCM, p < 0.0001). Histology revealed cardiomyocyte hypertrophy (cross-sectional area 393.2 ± 14.5 µm2 vs. 538.9 ± 23.8 µm2, p < 0.001) without fibrosis. Mitochondria were shifted to the intercalated discs and enlarged. Mitochondrial membrane potential remained stable in TCM. The metabolite profiles of ELVD and TCM were characterised by profound depletion of tricarboxylic acid cycle intermediates. Redox balance was shifted towards a more oxidised state (ratio of reduced to oxidised nicotinamide adenine dinucleotide 10.5 ± 2.1 vs. 4.0 ± 0.8, p < 0.01). The mitochondrial acetylome remained largely unchanged. Neither TCM nor TACH-iPSC-CM showed relevantly increased levels of reactive oxygen species. Oxidative phosphorylation capacity of TCM decreased modestly in skinned fibres (168.9 ± 11.2 vs. 124.6 ± 11.45 pmol·O2·s-1·mg-1 tissue, p < 0.05), but it did not in isolated mitochondria. The pattern of mitochondrial dysfunctions detected in two models of tachycardiomyopathy diverges from previously published characteristic signs of other heart failure aetiologies.

Determination of the effective dosage of tiletamine-zolazepam-ketamine-xylazine, with or without methadone, in dogs.

OBJECTIVE: To determine the effective dosage of the combination tiletamine-zolazepam-ketamine-xylazine (TKX), with or without methadone, in dogs. STUDY DESIGN: Prospective, randomized, experimental study. ANIMALS: A total of 29 dogs. METHODS: Dogs were randomly administered TKX (group TKX, n = 13) or combined with 0.3 mg kg-1 of methadone (group TKXM, n = 16) intramuscularly. The TKX solution contained tiletamine (50 mg mL-1), zolazepam (50 mg mL-1), ketamine (80 mg mL-1) and xylazine (20 mg mL-1). The effective dosages for immobility in 50% and 95% of the population (ED50 and ED95) were estimated using the up-and-down method. Approximately 20 minutes after drug administration, a skin incision was performed and the response was judged as positive or negative if the dogs moved or did not move, respectively. The TKX volume for the subsequent dog in the same group was increased or decreased by 0.005 mL kg-1 if the response of the previous dog was positive or negative, respectively. Heart and respiratory rates, and sedation/anesthesia scores (range 0-21) were recorded before and 15 minutes after drug administration. RESULTS: Estimated ED50 and ED95 (95% confidence intervals) were: TKX, 0.025 (0.020-0.029) and 0.026 (0.010-0.042) mL kg-1; TKXM, 0.022 (0.018-0.025) and 0.033 (0.017-0.049) mL kg-1. Median (interquartile range) scores for sedation/anesthesia were 17 (16-18) and 17 (15-20), and times until lateral recumbency were 5 (4-6) and 6 (4-10) minutes in TKX and TKXM, respectively (p > 0.05). In both groups heart and respiratory rates decreased, but values remained acceptable for anesthetized dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The results provide a guide for volumes of TKX and TKXM in dogs requiring restraint for minimally invasive procedures. Inclusion of methadone in the TKX combination did not influence ED50.

Epidural administration of combinations of ropivacaine, morphine and xylazine in bitches undergoing total unilateral mastectomy: a randomized clinical trial.

OBJECTIVE: To investigate the epidural administration of combinations of ropivacaine, morphine and xylazine in bitches undergoing unilateral mastectomy. STUDY DESIGN: Prospective, randomized, blinded, clinical study. ANIMALS: A total of 22 bitches scheduled to undergo unilateral mastectomy for mammary tumor excision. METHODS: Dogs were anesthetized with acepromazine (0.02 mg kg-1) and morphine (0.3 mg kg-1) intramuscularly, propofol intravenously (IV) and isoflurane. Prior to the beginning of surgery, dogs were randomly administered one of three epidural treatments: ropivacaine (0.75 mg kg-1) with morphine (0.1 mg kg-1) (group RM, n = 7); ropivacaine with xylazine (0.1 mg kg-1) (group RX, n = 8); or ropivacaine with morphine and xylazine (group RMX, n = 7). Cardiopulmonary variables and the expired concentration of isoflurane (Fe'Iso) were recorded intraoperatively. Meloxicam (0.1 mg kg-1) was administered IV during skin closure. Postoperative pain scores were evaluated with the Glasgow composite measure pain scale short form for 24 hours, and rescue analgesia with morphine (0.5 mg kg-1) was administered intramuscularly when pain scores were ≥ 6/24. RESULTS: Fe'Iso was significantly higher in group RM than in groups RX and RMX. Heart rate decreased significantly in groups RX and RMX, but blood pressure remained within acceptable values. The number of dogs administered rescue analgesia within 24 hours was significantly higher in group RX (seven dogs, 87.5%) than in groups RM (one dog, 14.3%; p = 0.01) and RMX (two dogs, 28.6%; p = 0.04). Time to standing was significantly longer in group RX than in group RM. CONCLUSIONS AND CLINICAL RELEVANCE: All epidural treatments provided adequate antinociception with minimal cardiovascular adverse effects during mastectomy. The inclusion of morphine (groups RM and RMX) provided the best postoperative analgesia. Owing to the undesirable effect of xylazine on ambulation, the combination ropivacaine-morphine appeared to provide greater benefits in bitches undergoing unilateral mastectomy.

Cardiac Fibrosis Is a Risk Factor for Severe COVID-19.

Increased left ventricular fibrosis has been reported in patients hospitalized with coronavirus disease 2019 (COVID-19). It is unclear whether this fibrosis is a consequence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection or a risk factor for severe disease progression. We observed increased fibrosis in the left ventricular myocardium of deceased COVID-19 patients, compared with matched controls. We also detected increased mRNA levels of soluble interleukin-1 receptor-like 1 (sIL1-RL1) and transforming growth factor ß1 (TGF-ß1) in the left ventricular myocardium of deceased COVID-19 patients. Biochemical analysis of blood sampled from patients admitted to the emergency department (ED) with COVID-19 revealed highly elevated levels of TGF-ß1 mRNA in these patients compared to controls. Left ventricular strain measured by echocardiography as a marker of pre-existing cardiac fibrosis correlated strongly with blood TGF-ß1 mRNA levels and predicted disease severity in COVID-19 patients. In the left ventricular myocardium and lungs of COVID-19 patients, we found increased neuropilin-1 (NRP-1) RNA levels, which correlated strongly with the prevalence of pulmonary SARS-CoV-2 nucleocapsid. Cardiac and pulmonary fibrosis may therefore predispose these patients to increased cellular viral entry in the lung, which may explain the worse clinical outcome observed in our cohort. Our study demonstrates that patients at risk of clinical deterioration can be identified early by echocardiographic strain analysis and quantification of blood TGF-ß1 mRNA performed at the time of first medical contact.

Phosphorylation of RyR2 Ser-2814 by CaMKII mediates ß1-adrenergic stress induced Ca2+ -leak from the sarcoplasmic reticulum.

Adrenergic stimulation, while being the central mechanism of cardiac positive inotropy, is a universally acknowledged inductor of undesirable sarcoplasmic reticulum (SR) Ca2+ leak. However, the exact mechanisms for this remained unspecified so far. This study shows that Ca2+ /calmodulin-dependent protein kinase II (CaMKII)-specific phosphorylation of ryanodine receptor type 2 at Ser-2814 is the pivotal mechanism by which SR Ca2+ leak develops downstream of ß1-adrenergic stress by increase of the leak/load relationship. Cardiomyocytes with a Ser-2814 phosphoresistant mutation (S2814A) were protected from isoproterenol-induced SR Ca2+ leak and consequently displayed improved postrest potentiation of systolic Ca2+ release under adrenergic stress compared to littermate wild-type cells.

Loss of CASK Accelerates Heart Failure Development.

[Figure: see text].

Decreased GLUT1/NHE1 RNA expression in whole blood predicts disease severity in patients with COVID-19.

AIMS: We aimed to assess whether expression of whole-blood RNA of sodium proton exchanger 1 (NHE1) and glucose transporter 1 (GLUT1) is associated with COVID-19 infection and outcome in patients presenting to the emergency department with respiratory infections. Furthermore, we investigated NHE1 and GLUT1 expression in the myocardium of deceased COVID-19 patients. METHODS AND RESULTS: Whole-blood quantitative assessment of NHE1 and GLUT1 RNA was performed using quantitative PCR in patients with respiratory infection upon first contact in the emergency department and subsequently stratified by SARS-CoV-2 infection status. Assessment of NHE1 and GLUT1 RNA using PCR was also performed in left ventricular myocardium of deceased COVID-19 patients. NHE1 expression is up-regulated in whole blood of patients with COVID-19 compared with other respiratory infections at first medical contact in the emergency department (control: 0.0021 ± 0.0002, COVID-19: 0.0031 ± 0.0003, P = 0.01). The ratio of GLUT1 to NHE1 is significantly decreased in the blood of COVID-19 patients who are subsequently intubated and/or die (severe disease) compared with patients with moderate disease (moderate disease: 0.497 ± 0.083 vs. severe disease: 0.294 ± 0.0336, P = 0.036). This ratio is even further decreased in the myocardium of patients who deceased from COVID-19 in comparison with the myocardium of non-infected donors. CONCLUSIONS: NHE1 and GLUT1 may be critically involved in the disease progression of SARS-CoV-2 infection. We show here that SARS-CoV-2 infection critically disturbs ion channel expression in the heart. A decreased ratio of GLUT1/NHE1 could potentially serve as a biomarker for disease severity in patients with COVID-19.

The oral Ca/calmodulin-dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure.

AIMS: Excessive activation of Ca/calmodulin-dependent kinase II (CaMKII) is of critical importance in heart failure (HF) and atrial fibrillation. Unfortunately, lack of selectivity, specificity, and bioavailability have slowed down development of inhibitors for clinical use. We investigated a novel CaMKIIδ/CaMKIIÉ£-selective, ATP-competitive, orally available CaMKII inhibitor (RA608) on right atrial biopsies of 119 patients undergoing heart surgery. Furthermore, we evaluated its oral efficacy to prevent deterioration of HF in mice after transverse aortic constriction (TAC). METHODS AND RESULTS: In human atrial cardiomyocytes and trabeculae, respectively, RA608 significantly reduced sarcoplasmic reticulum Ca leak, reduced diastolic tension, and increased sarcoplasmic reticulum Ca content. Patch-clamp recordings confirmed the safety of RA608 in human cardiomyocytes. C57BL6/J mice were subjected to TAC, and left ventricular function was monitored by echocardiography. Two weeks after TAC, RA608 was administered by oral gavage for 7 days. Oral RA608 treatment prevented deterioration of ejection fraction. At 3 weeks after TAC, ejection fraction was 46.1 ± 3.7% (RA608) vs. 34.9 ± 2.6% (vehicle), n = 9 vs. n = 12, P < 0.05, ANOVA, which correlated with significantly less CaMKII autophosphorylation at threonine 287. Moreover, a single oral dose significantly reduced inducibility of atrial and ventricular arrhythmias in CaMKIIδ transgenic mice 4 h after administration. Atrial fibrillation was induced in 6/6 mice for vehicle vs. 1/7 for RA608, P < 0.05, 'n - 1' χ2 test. Ventricular tachycardia was induced in 6/7 for vehicle vs. 2/7 for RA608, P < 0.05, 'n - 1' χ2 test. CONCLUSIONS: RA608 is the first orally administrable CaMKII inhibitor with potent efficacy in human myocytes. Moreover, oral administration potently inhibits arrhythmogenesis and attenuates HF development in mice in vivo.

Ca2+/calmodulin-dependent protein kinase II is essential in hyperacute pressure overload.

BACKGROUND: Activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is established as a central intracellular trigger for various cardiac pathologies such as hypertrophy, heart failure or arrhythmias in animals and humans suggesting CaMKII as a promising target protein for future medical treatments. However, the physiological role of CaMKII is scarcely well defined. AIM & METHODS: To investigate the role of CaMKII in hyperacute pressure overload, we evaluated the effects of pressure overload induced by transverse aortic constriction (TAC) on survival, cardiac function, protein expression and excitation-contraction coupling (ECC) in female WT littermate vs. AC3-I mice 2 days after TAC (2d post TAC). AC3-I mice express the CaMKII inhibitor autocamtide-3 related inhibitory peptide (AiP) under the control of the α-myosin heavy chain promotor in the heart. RESULTS: CaMKII activation is significantly increased in WT TAC vs. sham mice 2d post TAC. Interestingly, survival is significantly reduced in AC3-I animals within the first five days after TAC compared to WT TAC littermates, while systolic cardiac function is markedly reduced in AC3-I TAC vs. AC3-I sham mice, but preserved in WT TAC vs. WT sham mice. Proteins regulating ECC such as ryanodine receptors (RyR2) and phospholamban (PLB) are hypophosphorylated at their CaMKII phosphorylation site in AC3-I TAC mice, but hyperphosphorylated in WT TAC mice compared to controls. In isolated cardiomyocytes fractional shortening is significantly impaired in AC3-I compared to WT mice 2d post TAC, and CaMKII incubation with AiP mimics the AC3-I phenotype in cardiomyocytes from WT TAC mice in vitro. In summary, this suggests cardiac dysfunction due to CaMKII inhibition as a potential cause of increased mortality in AC3-I TAC mice. However, proarrhythmic spontaneous Ca2+ release events (SCR) appear less frequent in cardiomyocytes from AC3-I TAC mice than in WT TAC mice. CONCLUSIONS: Our data indicate that excessive CaMKII inhibition as present in AC3-I transgenic mice leads to an impaired adaptation of ECC to hyperacute pressure overload resulting in diminished cardiac contractility and increased death. Thus, our data suggest that in pressure overload the activation of CaMKII is a pivotal, but previously unknown part of hyperacute stress physiology in the heart, while CaMKII inhibition, albeit potentially antiarrhythmic, can be detrimental. This should be taken into account for future studies with CaMKII inhibitors as therapeutic agents.

Subzygomatic and infraorbital approaches for maxillary nerve blockade in cats' cadaver / Abordagens subzigomática e infraorbitária para o bloqueio do nervo maxilar em cadáver de gatos

ABSTRACT: This study compared the accuracy of dye placement on the maxillary nerve by using the percutaneous subzigomatic (SBZ) and infraorbitary (IO) approaches in cats' cadavers. A second aim was to compare the accuracy of dye placement on the maxillary nerve between different untrained anesthetists. This was a prospective, randomized, blinded study, performed in 40 heads obtained from feline cadavers. Three veterinarians (A, B and C) with no previous experience with the IO approach performed the experiments. The SBZ approach was randomly performed on one side of the head and the IO approach was performed in the contralateral side of the same head. For each approach, 0.2ml of 1% methylene blue dye was injected. Scores for length of nerve staining were as follows: 0 (failure), no staining; 1 (moderate), <6mm of nerve stained; and 2 (ideal), ≥6mm of nerve stained. Median scores (interquartile range) for the SBZ and IO approaches were 2.0 (0.3-2.0) and 1.0 (0.0-2.0), respectively. Scores for length of nerve staining were higher with the SBZ approach than the IO approach (P=0.016). Considering the scores for both the SBZ and IO approaches, there was a significant difference among the three veterinarians (P=0.002). Results of this study do not support the IO approach to perform a maxillary nerve block in cats. A greater accuracy of methylene blue dye placement was observed with the SBZ approach. A variable accuracy may exist between different veterinarians when performing a maxillary nerve block employing the SBZ and IO techniques in cats.

RESUMO: O objetivo deste estudo foi comparar o acesso do nervo maxilar pela abordagem subzigomática (SBZ) com a abordagem pelo forame infraorbitário (IO) em peças anatômicas de gatos utilizando o corante azul de metileno. Um segundo objetivo foi comparar a acurácia na coloração do nervo maxilar com o azul de metileno entre diferentes anestesistas que não receberam treinamento prévio. Este estudo foi prospectivo, randomizado, cego, realizado em 40 peças anatômicas de cabeças de gatos. Três veterinários (A, B e C), sem experiência prévia da abordagem IO, realizaram o experimento. A abordagem SBZ foi aleatoriamente realizada em um dos lados da cabeça e a abordagem IO foi realizada no lado contralateral da mesma peça anatômica. Para cada abordagem, utilizou-se 0,2mL do corante azul de metileno 1%. Classificou-se o escore de coloração baseado no comprimento do nervo maxilar corado pelo azul de metileno conforme a escala: 0 (falha da técnica), sem coloração; 1 (moderado), <6mm de coloração do nervo maxilar; 2 (ideal), ≥6mm de coloração do nervo maxilar. As medianas (intervalo interquartil) para as abordagens SBZ e IO (dados de todos os veterinários juntos) foram respectivamente 2,0 (0,3-2,0) e 1,0 (0,0-2,0). A abordagem SBZ foi associada a um escore de coloração, significativamente, maior do que a abordagem IO (P=0,016). Considerando os escores de ambas abordagens (SBZ e IO), houve diferença significativa nos escores de coloração do nervo maxilar entre os três veterinários anestesistas (P=0,002). Os resultados deste estudo não sustentam a utilização da abordagem IO para a realização do bloqueio maxilar em gatos. Uma melhor acurácia na coloração do nervo maxilar com o azul de metileno foi observada com a abordagem SBZ. A acurácia da técnica pode variar quando as abordagens SBZ e IO são realizadas por veterinários diferentes, com o objetivo de se obter o bloqueio do nervo maxilar.

Mutation Spectrum of the ABCA4 Gene in 335 Stargardt Disease Patients From a Multicenter German Cohort-Impact of Selected Deep Intronic Variants and Common SNPs.

Purpose: Stargardt disease (STGD1) is an autosomal recessive retinopathy, caused by mutations in the retina-specific ATP-binding cassette transporter (ABCA4) gene. To establish the mutational spectrum and to assess effects of selected deep intronic and common genetic variants on disease, we performed a comprehensive sequence analysis in a large cohort of German STGD1 patients. Methods: DNA samples of 335 STGD1 patients were analyzed for ABCA4 mutations in its 50 coding exons and adjacent intronic sequences by resequencing array technology or next generation sequencing (NGS). Parts of intron 30 and 36 were screened by Sanger chain-terminating dideoxynucleotide sequencing. An in vitro splicing assay was used to test selected variants for their splicing behavior. By logistic regression analysis we assessed the association of common ABCA4 alleles while a multivariate logistic regression model calculated a genetic risk score (GRS). Results: Our analysis identified 148 pathogenic or likely pathogenic mutations, of which 48 constitute so far unpublished ABCA4-associated disease alleles. Four rare deep intronic variants were found once in 472 alleles analyzed. In addition, we identified six risk-modulating common variants. Genetic risk score estimates suggest that defined common ABCA4 variants influence disease risk in carriers of a single pathogenic ABCA4 allele. Conclusions: Our study adds to the mutational spectrum of the ABCA4 gene. Moreover, in our cohort, deep intronic variants in intron 30 and 36 likely play no or only a minor role in disease pathology. Of note, our findings demonstrate a possible modifying effect of common sequence variants on ABCA4-associated disease.