Journal of molecular and cellular cardiology plus最新文献

An increase in mitochondrial calcium via the mitochondrial calcium uniporter (MCU) has been implicated in initiating cell death in the heart during ischemia-reperfusion (I/R) injury. Measurement of calcium during I/R has been challenging due to the pH sensitivity of indicators coupled with the fall in pH during I/R. The development of a pH-insensitive indicator, mitochondrial localized Turquoise Calcium Fluorescence Lifetime Sensor (mito-TqFLITS), allows for quantifying mitochondrial calcium during I/R via fluorescent lifetime imaging. Mitochondrial calcium was monitored using mito-TqFLITS in neonatal mouse ventricular myocytes (NMVM) isolated from germline MCU-KO mice and MCU^fl/fl treated with CRE-recombinase to acutely knockout MCU. To simulate ischemia, a coverslip was placed on a monolayer of NMVMs to prevent access to oxygen and nutrients. Reperfusion was induced by removing the coverslip. Mitochondrial calcium increases threefold during coverslip hypoxia in MCU-WT. There is a significant increase in mitochondrial calcium during coverslip hypoxia in germline MCU-KO, but it is significantly lower than in MCU-WT. We also found that compared to WT, acute MCU-KO resulted in no difference in mitochondrial calcium during coverslip hypoxia and reoxygenation. To determine the role of mitochondrial calcium uptake via MCU in initiating cell death, we used propidium iodide to measure cell death. We found a significant increase in cell death in both the germline MCU-KO and acute MCU-KO, but this was similar to their respective WTs. These data demonstrate the utility of mito-TqFLITS to monitor mitochondrial calcium during simulated I/R and further show that germline loss of MCU attenuates the rise in mitochondrial calcium during ischemia but does not reduce cell death.

Type 2 diabetes mellitus (T2DM) is a metabolic disease and comorbidity associated with several conditions, including cardiac dysfunction leading to heart failure with preserved ejection fraction (HFpEF), in turn resulting in T2DM-induced cardiomyopathy (T2DM-CM). However, the molecular mechanisms underlying the development of T2DM-CM are poorly understood. It is hypothesized that molecular alterations in myopathic genes induced by diabetes promote the development of HFpEF, whereas cardiac myosin inhibitors can rescue the resultant T2DM-mediated cardiomyopathy. To test this hypothesis, a Leptin receptor-deficient db/db homozygous (Lepr db/db) mouse model was used to define the pathogenesis of T2DM-CM. Echocardiographic studies at 4 and 6 months revealed that Lepr db/db hearts started developing cardiac dysfunction by four months, and left ventricular hypertrophy with diastolic dysfunction was evident at 6 months. RNA-seq data analysis, followed by functional enrichment, revealed the differential regulation of genes related to cardiac dysfunction in Lepr db/db heart tissues. Strikingly, the level of cardiac myosin binding protein-C phosphorylation was significantly increased in Lepr db/db mouse hearts. Finally, using isolated skinned papillary muscles and freshly isolated cardiomyocytes, CAMZYOS® (mavacamten, MYK-461), a prescription heart medicine used for symptomatic obstructive hypertrophic cardiomyopathy treatment, was tested for its ability to rescue T2DM-CM. Compared with controls, MYK-461 significantly reduced force generation in papillary muscle fibers and cardiomyocyte contractility in the db/db group. This line of evidence shows that 1) T2DM-CM is associated with hyperphosphorylation of cardiac myosin binding protein-C and 2) MYK-461 significantly lessened disease progression in vitro, suggesting its promise as a treatment for HFpEF.

Introduction

Hypertrophic cardiomyopathy (HCM) results from pathogenic variants in sarcomeric protein genes that increase myocyte energy demand and lead to cardiac hypertrophy. However, it is unknown whether a common metabolic trait underlies cardiac phenotype at the early disease stage. To address this question and define cardiac biochemical pathology in early-stage HCM, we studied two HCM mouse models that express pathogenic variants in cardiac troponin T (Tnt2) or myosin heavy chain (Myh6) genes, and have marked differences in cardiac imaging phenotype, mitochondrial function at early disease stage.

Methods

We used a combination of echocardiography, transcriptomics, mass spectrometry-based untargeted metabolomics (GC-TOF, HILIC, CSH-QTOF), and computational modeling (CardioNet) to examine cardiac structural and metabolic remodeling at early disease stage (5 weeks of age) in R92W-TnT^+/− and R403Q-MyHC^+/− mutant mice. Data from mutants was compared with respective littermate controls (WT).

Results

Allele-specific differences in cardiac phenotype, gene expression and metabolites were observed at early disease stage. LV diastolic dysfunction was prominent in TnT mutants. Differentially-expressed genes in TnT mutant hearts were predominantly enriched in the Krebs cycle, respiratory electron transport, and branched-chain amino acid metabolism, whereas MyHC mutants were enriched in mitochondrial biogenesis, calcium homeostasis, and liver-X-receptor signaling. Both mutant hearts demonstrated significant alterations in levels of purine nucleosides, trisaccharides, dicarboxylic acids, acylcarnitines, phosphatidylethanolamines, phosphatidylinositols, ceramides and triglycerides; 40.4 % of lipids and 24.7 % of metabolites were significantly different in TnT mutants, whereas 10.4 % of lipids and 5.8 % of metabolites were significantly different in MyHC mutants. Both mutant hearts had a lower abundance of unsaturated long-chain acyl-carnitines (18:1, 18:2, 20:1), but only TnT mutants showed enrichment of FA18:0 in ceramide and cardiolipin species. CardioNet predicted impaired energy substrate metabolism and greater phospholipid remodeling in TnT mutants than in MyHC mutants.

Conclusions

Our systems biology approach revealed marked differences in metabolic remodeling in R92W-TnT and R403Q-MyHC mutant hearts, with TnT mutants showing greater derangements than MyHC mutants, at early disease stage. Changes in cardiolipin composition in TnT mutants could contribute to impairment of energy metabolism and diastolic dysfunction observed in this study, and predispose to energetic stress, ventricular arrhythmias under high workloads such as exercise.

Background

Pulmonary hypertension (PH) often leads to right ventricle (RV) failure, a significant cause of morbidity and mortality. Despite advancements in PH management, progression to RV maladaptation and subsequent failure remain a clinical challenge. This study explored the effect of paroxetine, a selective serotonin reuptake inhibitor (SSRI), on RV function in a rat model of PH, hypothesizing that it improves RV function by inhibiting G protein-coupled receptor kinase 2 (GRK2) and altering myofilament protein phosphorylation.

Methods

The Su5416/hypoxia (SuHx) rat model was used to induce PH. Rats were treated with paroxetine and compared to vehicle-treated and control groups. Parameters measured included RV morphology, systolic and diastolic function, myofilament protein phosphorylation, GRK2 activity, and sympathetic nervous system (SNS) markers.

Results

Paroxetine treatment significantly improved RV systolic function, evidenced by increased stroke volume, cardiac output, and ejection fraction, without significantly affecting RV hypertrophy, myosin heavy chain/titin isoform switching, or fibrosis. Enhanced phosphorylation of titin and myosin light chain-2 was observed, correlating positively with improved systolic function. Contrary to the hypothesis, improvements occurred independently of GRK2 inhibition or SNS modulation, suggesting an alternate mechanism, potentially involving antioxidant properties of paroxetine.

Conclusion

Paroxetine improves RV systolic function in PH rats, likely through mechanisms beyond GRK2 inhibition, possibly related to its antioxidant effects. This highlights the potential of paroxetine in managing RV dysfunction in PH, warranting further investigation into its detailed mechanisms of action and clinical applicability.

Few immortalized cardiac microvascular endothelial cell (CMEC) lines are available, particularly mouse lines. We purchased the CLU510 mCMEC line (Cedarlane), isolated by fluorescence-activated cell sorting for CD31 and VE-cadherin. The cell line has been used in previous studies, although none report CD31 or VE-cadherin expression. We analyzed endothelial profile of two vials of passage 38 cells. CD31 and VE-cadherin mRNA were hardly expressed in mCMECs compared to primary mouse lung ECs. CD31 and VE-cadherin protein levels were also negligible compared to multiple EC lines. Thus, CLU510 mCMECs beyond P38 do not harbor an endothelial phenotype. Caution should be warranted when using commercial cells and journals should carefully consider the validity of results when essential characterization of cell lines is omitted.

Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvβ3-specific RGD-mimetic, 29P, was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 μg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvβ3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.

KairoSight-3.0 is a recently released Python-based, open-source software for cardiac optical mapping analysis. Addressing challenges in high-resolution electrophysiological data analysis, KairoSight-3.0 facilitates comprehensive studies of cardiac conduction and excitation-contraction coupling. We compared its performance with ElectroMap, focusing on action potential duration and conduction velocity measurements in mouse heart models subjected to ischaemia and flecainide treatment. Our findings reveal that while both software are effective, inherent methodological differences impact measurement outcomes. KairoSight-3.0's robust analysis capabilities make it a valuable tool in cardiac research. Additionally, future directions for KairoSight-3.0 and other mapping analysis tools are explored.

Statement of importance

Open-source methods for analysis of cardiac optical mapping are vital tools in electrophysiological research. Our work directly evaluates the latest version of KarioSight, recently published in JMCC plus, with ElectroMap, an established and widely used tool. Our results show both software are effective in analysis of changes in both conduction and repolarisation. Considering the new features of KairoSight-3.0 and python implementation, our study importantly demonstrates the effectiveness of the software, highlights potential discrepancies between it and ElectroMap, and provides a perspective on future directions for KairoSight-3.0 and other software.

Cardiac microtubules have recently been implicated in mechanical dysfunction during heart failure. However, systemic intolerance and non-cardiac effects of microtubule-depolymerizing compounds have made it challenging to determine the effect of microtubules on myocardial performance. Herein, we leverage recent advancements in living myocardial slices to develop a stable working preparation that recapitulates the complexity of diastole by including early and late phases of diastolic filling. To determine the effect of cardiac microtubule depolymerization on diastolic performance, myocardial slices were perfused with oxygenated media to maintain constant isometric twitch forces for more than 90 min. Force-length work loops were collected before and after 90 min of treatment with either DMSO (vehicle) or colchicine (microtubule depolymerizer). A trapezoidal stretch was added prior to the beginning of ventricular systole to mimic late-stage-diastolic filling driven by atrial systole. Force-length work loops were obtained at fixed preload and afterload, and tissue velocity was obtained during diastole as an analog to trans-mitral Doppler. In isometric twitches, microtubule destabilization accelerated force development, relaxation kinetics, and decreased end diastolic stiffness. In work loops, microtubule destabilization increased stroke length, myocardial output, accelerated isometric contraction and relaxation, and increased the amplitude of early filling. Taken together, these results indicate that the microtubule destabilizer colchicine can improve diastolic performance by accelerating isovolumic relaxation and early filling leading to increase in myocardial work output.

Mitochondrial (MITO) dysfunction occurs in the failing heart and contributes to worsening of heart failure (HF). Reduced aldehyde dehydrogenase 2 (ALDH2) in left ventricular (LV) myocardium of diabetic hearts has been implicated in MITO dysfunction through accumulation of toxic aldehydes including and elevated levels of 4-hydroxy-2-nonenal (4HNE). This study examined whether dysregulation of MITO ALDH2 (mALDH2) occurs in mitochondria of the failing LV and is associated with increased levels of 4HNE.

LV tissue from 7 HF and 7 normal (NL) dogs was obtained. Protein quantification of total mitochondrial ALDH2 (t-mALDH2), phosphorylated mALDH2 (p-mALDH2), total MITO protein kinase c epsilon (t-mPKCε), phosphorylated mPKCε (p-mPKCε) was performed by Western blotting, and total mALDH2 enzymatic activity was measured. Protein adducts of 4HNE-MITO and 4HNE-mALDH2 were also measured in MITO fraction by Western Blotting.

Protein level of t-mALDH2 was decreased in HF compared with NL dogs (0.63 ± 0.07 vs 1.17 ± 0.08, p < 0.05) as did mALDH2 enzymatic activity (51.39 ± 3 vs. 107.66 ± 4 nmol NADH/min/mg, p < 0.05). Phosphorylated-mALDH2 and p-mPKCε were unchanged. 4HNE-MITO proteins adduct levels increased in HF compared with NL (2.45 ± 0.08 vs 1.30 ± 0.03 du, p < 0.05) as did adduct levels of 4HNE-mALDH2 (1.60 ± 0.20 vs 0.39 ± 0.08, p < 0.05). In isolated failing cardiomyocytes (CM) exposure to 4HNE decreased mALDH2 activity, increased ROS and 4HNE-ALDH2 adducts, and worsened MITO function. Stimulation of mALDH2 activity with ALDA-1 in isolated HF CMs compared to NL CMs improved ADP-stimulated respiration and maximal ATP synthesis to a greater extant (+47 % and +89 %, respectively).

Down-regulation of mALDH2 protein levels and activity occurs in HF and contributes to MITO dysfunction and is likely caused by accumulation of 4HNE-mALDH2 adduct. Increasing mALDH2 activity (via ALDA-1) improved MITO function in failing CMs.

Background

In persistent atrial fibrillation (AF), localized extra-pulmonary vein sources may contribute to arrhythmia recurrences after pulmonary vein isolation. This in-silico study proposes a high-density sequential mapping strategy to localize such sources.

Method

Catheter repositioning was guided by repetitive conduction patterns, moving against the prevailing conduction direction (upstream) toward the sources. Sources were found either by locally identifying conduction patterns or by encircling the region harboring them. We simulated source tracking in an in-silico atrial model, comparing random vs. upstream-guided catheter repositioning (with and without encircling). To assess performance in increasing AF complexities, we simulated AF in 3 groups: atria with reentry-anchoring scars, without fibrosis, and with severe endomysial fibrosis.

Results

Compared to random mapping, the upstream-guided approach successfully located sources more often (anchored reentries: $70.6\%$ vs. $10.6\%$; no fibrosis: $87.9\%$ vs. $22.1\%$; with fibrosis: $95.0\%$ vs. $60.9\%$ of tracking procedures, all $p<0.001$), using fewer steps (median [IQR]: 11 [7;23] vs. 26 [13;35]; 10 [6;19] vs. 19 [10;27]; 11 [7;19] vs. 16 [8;30], respectively, all $p<0.05$). Adding source encircling increased source detection (98.1 %, 100 %, and 99.5 %, all $p<0.01$ vs. local detection only), reducing required steps (9 [6;12], 8 [6;12], and 9 [6;13], all $p<0.05$). In some cases (11.9 %, 17.1 %, and 10.5 % of procedures), the algorithm encircled regions $>$15 mm from the source.

Conclusion

Moving mapping catheters upstream improves source detection efficiency, even in the presence of severe fibrosis. Encircling sources may help find regions of interest in fewer steps.