MicroRNA-365 regulates human cardiac action potential duration

Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current Iks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.

A left atrial hamartoma of mature cardiac myocytes

We present a case of a hamartoma of mature cardiac myocytes. This is an extremely rare tumour and the first reported paediatric case localised in the left atrium.

Effects of tamoxifen inducible MerCreMer on gene expression in cardiac myocytes in mice

The Cre-LoxP technology, including the tamoxifen (TAM) inducible MerCreMer (MCM), is increasingly used to delineate gene function, understand the disease mechanisms, and test therapeutic interventions. We set to determine the effects of TAM-MCM on cardiac myocyte transcriptome. Expression of the MCM was induced specifically in cardiac myocytes upon injection of TAM to myosin heavy chain 6-MCM (Myh6-Mcm) mice for 5 consecutive days. Cardiac function, myocardial histology, and gene expression (RNA-sequencing) were analyzed 2 weeks after TAM injection. A total of 346 protein coding genes (168 up- and 178 down-regulated) were differentially expressed. Transcript levels of 85 genes, analyzed by a reverse transcription-polymerase chain reaction in independent samples, correlated with changes in the RNA-sequencing data. The differentially expressed genes were modestly enriched for genes involved in the interferon response and the tumor protein 53 (TP53) pathways. The changes in gene expression were relatively small and mostly transient and had no discernible effects on cardiac function, myocardial fibrosis, and apoptosis or induction of double-stranded DNA breaks. Thus, TAM-inducible activation of MCM alters cardiac myocytes gene expression, provoking modest and transient interferon and DNA damage responses without exerting other discernible phenotypic effects. Thus, the effects of TAM-MCM on gene expression should be considered in discerning the bona fide changes that result from the targeting of the gene of interest.

Histopathological Features and Protein Markers of Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is a heritable heart muscle disease characterized by syncope, palpitations, ventricular arrhythmias and sudden cardiac death (SCD) especially in young individuals. It is estimated to affect 1:5,000 individuals in the general population, with >60% of patients bearing one or more mutations in genes coding for desmosomal proteins. Desmosomes are intercellular adhesion junctions, which in cardiac myocytes reside within the intercalated disks (IDs), the areas of mechanical and electrical cell-cell coupling. Histologically, ACM is characterized by fibrofatty replacement of cardiac myocytes predominantly in the right ventricular free wall though left ventricular and biventricular forms have also been described. The disease is characterized by age-related progression, vast phenotypic manifestation and incomplete penetrance, making proband diagnosis and risk stratification of family members particularly challenging. Key protein redistribution at the IDs may represent a specific diagnostic marker but its applicability is still limited by the need for a myocardial sample. Specific markers of ACM in surrogate tissues, such as the blood and the buccal epithelium, may represent a non-invasive, safe and inexpensive alternative for diagnosis and cascade screening. In this review, we shall cover the most relevant biomarkers so far reported and discuss their potential impact on the diagnosis, prognosis and management of ACM.

Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes

It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart’s biology in health and disease.

Loss of Mitochondrial Ca 2+ Uniporter Limits Inotropic Reserve and Provides Trigger and Substrate for Arrhythmias in Barth Syndrome Cardiomyopathy

Background: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Since mitochondrial function and ROS formation are regulated by excitation-contraction (EC) coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. Methods: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin ( Taz -KD) compared to wild-type (WT) littermates. Respiratory chain assembly and function, ROS emission, and Ca 2+ uptake were determined in isolated mitochondria. EC coupling was integrated with mitochondrial redox state, ROS, and Ca 2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo . Results: Taz -KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca 2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, partly compensated by accelerated diastolic Ca 2+ decay through preactivated sarcoplasmic reticulum Ca 2+ ATPase (SERCA). Taz deficiency provoked heart-specific loss of mitochondrial Ca 2+ uniporter (MCU) protein that prevented Ca 2+ -induced activation of the Krebs cycle during β-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo , Taz -KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to β-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca 2+ export via the mitochondrial Na + /Ca 2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo . Conclusions: Downregulation of MCU, increased myofilament Ca 2+ affinity, and preactivated SERCA provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca 2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.

Altered Cardiolipin Metabolism is Associated with Cardiac Mitochondrial Dysfunction in Pulmonary Vascular Remodeled Perinatal Rat Pups

BackgroundPulmonary vascular remodeling (PVR) in utero results in the development of heart failure (HF). The alterations that occur in cardiac lipid and mitochondrial bioenergetics during the development of in utero PVR was unknown.MethodsPVR was induced in pups in utero by exposure of pregnant dams to indomethacin and hypoxia. Cardiac lipids, echocardiographic function and cardiomyocyte mitochondrial function were subsequently examined.ResultsPerinatal rat pups with PVR exhibited elevated left and right cardiac ventricular internal dimensions and reduced ejection fraction and fractional shortening compared to controls. Cardiac myocytes from these pups exhibited increased glycolytic capacity and glycolytic reserve compared to controls. However, respiration with glucose as substrate was unaltered. Fatty acid oxidation and ATP-insensitive respiration were increased in isolated cardiac myocytes from these pups compared to controls indicating mitochondrial dysfunction. Although abundance of mitochondrial respiratory complexes were unaltered, increased trilinoleoyl-lysocardiolipin levels in these pups was observed. A compensatory increase in both cardiolipin (CL) and phosphatidylethanolamine (PE) content were observed due to increased synthesis of these phospholipids.ConclusionAlterations in cardiac cardiolipin and phospholipid metabolism in PVR rat pups is associated with the mitochondrial bioenergetic and cardiac functional defects observed in their hearts.Impact statement- Phospholipid metabolism was examined in pulmonary vascular remodeling in perinatal rat pups.- Pulmonary vascular remodeling was induced in utero by treating pregnant dams with hypoxia and indomethacin at 19-21 days of gestation.- The offspring exhibited altered pulmonary arterial remodeling with subsequent cardiac hypertrophy, ventricular dysfunction, cardiac myocyte mitochondrial dysfunction with altered fatty acid utilization.- In addition, the offspring exhibited elevated cardiolipin, lysocardiolipin and phosphatidylethanolamine content which may potentially contribute to the cardiac mitochondrial dysfunction.

Expression and roles of N-type Ca channel in cardiaomyocytes

Background Voltage dependent Ca channels are divided to L-, T-, N-, P/Q-, and R-types, and N-type Ca channel (NCC) is mainly expressed in nerve terminal and regulates neurotransmitter release. Recently, NCC has been reported to express in adrenal gland and renal tubular cells. We examined whether NCC is expressed in cardiac myocytes and if so, the roles of this channel. Methods Expression of NCC mRNA and protein in cardiomyocytes were assessed by quantitative real time PCR and Western blot analysis using neonatal rat cultured cardiomyocytes, infant, and adult rat hearts. Expression site of NCC in cardiomyocytes was examined by confocal imaging of immunofluorescent staining. The roles of NCC in physiological Ca transient in neonatal myocytes were examined using fluorescence imaging of Fluo4, an intracellular Ca indicator. To examine the effects of pathological condition, such as heart failure and ischemia-reperfusion, on NCC expression, cultured cardiomyocytes were treated with norepinephrine (10 μmol/L, 24 hours) or subjected to 5 hours of hypoxia followed by 30 minutes of reoxygenation. In addition, adult rats were subjected to myocardial infarction by ligating the left anterior coronary artery. Lethal myocyte injury was examined by LDH activity in culture medium and myocyte apoptosis was examined by nuclear staining with DAPI and caspase 3 activity. To clarify the roles of NCC in neonatal myocytes in these pathological conditions, we examine the effect of ω-conotoxin, a selective NCC blocker. Results NCC mRNA and protein were expressed in neonatal cardiomyocytes. Immunocytochemical staining showed NCC was expressed in myocyte plasma membrane. During physiological spontaneous beating, ω-conotoxin did not affect beating rate and intra cellular Ca transient, suggesting that the roles of NCC on physiological beating are little. After birth level of NCC mRNA expression in cardiac tissue gradually decreased within 2 weeks and low level of mRNA expressed continuously in adult cardiac tissue. However, in pathological condition, mRNA and protein levels of NCC in non-infarcted region were increased 4 weeks after myocardial infarction. In addition, hypoxia-reoxygenation and norepinephrine administration increased LDH release and myocyte apoptosis in association with increase in NCC expression in neonatal cultured myocytes. ω-conotoxin significantly suppressed hypoxia/reoxygenation- and norepinephrine-induced LDH release and caspase 3 activation. Conclusion NCC is expressed in neonatal cardiac myocytes and the expression level was decreased after birth. Pathological condition, such as ischemic heart disease and heart failure, upregulated NCC expression in cardiomyocytes and NCC exacerbated lethal myocyte injury, while roles of NCC in physiological beating are little. FUNDunding Acknowledgement Type of funding sources: None.