Phosphorylation of cardiac sodium channel at Ser571 anticipates manifestations of the aging myopathy.

Diastolic dysfunction and delayed ventricular repolarization are typically observed in the elderly, but whether these defects are intimately associated with the progressive manifestation of the aging myopathy remains to be determined. In this regard, aging in experimental animals is coupled with increased late Na+ current (INa,L) in cardiomyocytes, raising the possibility that INa,L conditions the modality of electrical recovery and myocardial relaxation of the aged heart. For this purpose, aging male and female wild-type (WT) C57Bl/6 mice were studied together with genetically engineered mice with phosphomimetic (gain of function, GoF) or ablated (loss of function, LoF) mutations of the sodium channel Nav1.5 at Ser571 associated with, respectively, increased and stabilized INa,L. At ∼18 mo of age, WT mice developed prolonged duration of the QT interval of the electrocardiogram and impaired diastolic left ventricular (LV) filling, defects that were reversed by INa,L inhibition. Prolonged repolarization and impaired LV filling occurred prematurely in adult (∼5 mo) GoF mutant mice, whereas these alterations were largely attenuated in aging LoF mutant animals. Ca2+ transient decay and kinetics of myocyte shortening/relengthening were delayed in aged (∼24 mo) WT myocytes, with respect to adult cells. In contrast, delayed Ca2+ transients and contractile dynamics occurred at adult stage in GoF myocytes and further deteriorated in old age. Conversely, myocyte mechanics were minimally affected in aging LoF cells. Collectively, these results document that Nav1.5 phosphorylation at Ser571 and the late Na+ current modulate the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction.NEW & NOTEWORTHY We have investigated the impact of the late Na current (INa,L) on cardiac and myocyte function with aging by using genetically engineered animals with enhanced or stabilized INa,L, due to phosphomimetic or phosphoablated mutations of Nav1.5. Our findings support the notion that phosphorylation of Nav1.5 at Ser571 prolongs myocardial repolarization and impairs diastolic function, contributing to the manifestations of the aging myopathy.

Heart Rate Variability Reveals Altered Autonomic Regulation in Response to Myocardial Infarction in Experimental Animals.

The analysis of beating rate provides information on the modulatory action of the autonomic nervous system on the heart, which mediates adjustments of cardiac function to meet hemodynamic requirements. In patients with myocardial infarction, alterations of heart rate variability (HRV) have been correlated to the occurrence of arrhythmic events and all-cause mortality. In the current study, we tested whether experimental rodent models of myocardial infarction recapitulate dynamics of heart rate variability observed in humans, and constitute valid platforms for understanding mechanisms linking autonomic function to the development and manifestation of cardiovascular conditions. For this purpose, HRV was evaluated in two engineered mouse lines using electrocardiograms collected in the conscious, restrained state, using a tunnel device. Measurements were obtained in naïve mice and animals at 3-∼28 days following myocardial infarction, induced by permanent coronary artery ligation. Two mouse lines with inbred and hybrid genetic background and, respectively, homozygous (Homo) and heterozygous (Het) for the MerCreMer transgene, were employed. In the naïve state, Het female and male mice presented prolonged RR interval duration (∼9%) and a ∼4-fold increased short- and long-term RR interval variability, with respect to sex-matched Homo mice. These differences were abrogated by pharmacological interventions inhibiting the sympathetic and parasympathetic axes. At 3-∼14 days after myocardial infarction, RR interval duration increased in Homo mice, but was not affected in Het animals. In contrast, Homo mice had minor modifications in HRV parameters, whereas substantial (> 50%) reduction of short- and long-term RR interval variation occurred in Het mice. Interestingly, ex vivo studies in isolated organs documented that intrinsic RR interval duration increased in infarcted vs. non-infarcted Homo and Het hearts, whereas RR interval variation was not affected. In conclusion, our study documents that, as observed in humans, myocardial infarction in rodents is associated with alterations in heart rhythm dynamics consistent with sympathoexcitation and parasympathetic withdrawal. Moreover, we report that mouse strain is an important variable when evaluating autonomic function via the analysis of HRV.

Scn1b expression in the adult mouse heart modulates Na+ influx in myocytes and reveals a mechanistic link between Na+ entry and diastolic function.

Voltage-gated sodium channels (VGSCs) are macromolecular assemblies composed of a number of proteins regulating channel conductance and properties. VGSCs generate Na+ current (INa) in myocytes and play fundamental roles in excitability and impulse conduction in the heart. Moreover, VGSCs condition mechanical properties of the myocardium, a process that appears to involve the late component of INa. Variants in the gene SCN1B, encoding the VGSC ß1- and ß1B-subunits, result in inherited neurological disorders and cardiac arrhythmias. But the precise contributions of ß1/ß1B-subunits and VGSC integrity to the overall function of the adult heart remain to be clarified. For this purpose, adult mice with cardiac-restricted, inducible deletion of Scn1b (conditional knockout, cKO) were studied. Myocytes from cKO mice had increased densities of fast (+20%)- and slow (+140%)-inactivating components of INa, with respect to control cells. By echocardiography and invasive hemodynamics, systolic function was preserved in cKO mice, but diastolic properties and ventricular compliance were compromised, with respect to control animals. Importantly, inhibition of late INa with GS967 normalized left ventricular filling pattern and isovolumic relaxation time in cKO mice. At the cellular level, cKO myocytes presented delayed kinetics of Ca2+ transients and cell mechanics, defects that were corrected by inhibition of INa. Collectively, these results document that VGSC ß1/ß1B-subunits modulate electrical and mechanical function of the heart by regulating, at least in part, Na+ influx in cardiomyocytes.NEW & NOTEWORTHY We have investigated the consequences of deletion of Scn1b, the gene encoding voltage-gated sodium channel ß1-subunits, on myocyte and cardiac function. Our findings support the notion that Scn1b expression controls properties of Na+ influx and Ca2+ cycling in cardiomyocytes affecting the modality of cell contraction and relaxation. These effects at the cellular level condition electrical recovery and diastolic function in vivo, substantiating the multifunctional role of ß1-subunits in the physiology of the heart.

Diabetes-Induced Cellular Senescence and Senescence-Associated Secretory Phenotype Impair Cardiac Regeneration and Function Independently of Age.

Diabetes mellitus (DM) affects the biology of multipotent cardiac stem/progenitor cells (CSCs) and adult myocardial regeneration. We assessed the hypothesis that senescence and senescence-associated secretory phenotype (SASP) are main mechanisms of cardiac degenerative defect in DM. Accordingly, we tested whether ablation of senescent CSCs would rescue the cardiac regenerative/reparative defect imposed by DM. We obtained cardiac tissue from nonaged (50- to 64-year-old) patients with type 2 diabetes mellitus (T2DM) and without DM (NDM) and postinfarct cardiomyopathy undergoing cardiac surgery. A higher reactive oxygen species production in T2DM was associated with an increased number of senescent/dysfunctional T2DM-human CSCs (hCSCs) with reduced proliferation, clonogenesis/spherogenesis, and myogenic differentiation versus NDM-hCSCs in vitro. T2DM-hCSCs showed a defined pathologic SASP. A combination of two senolytics, dasatinib (D) and quercetin (Q), cleared senescent T2DM-hCSCs in vitro, restoring their expansion and myogenic differentiation capacities. In a T2DM model in young mice, diabetic status per se (independently of ischemia and age) caused CSC senescence coupled with myocardial pathologic remodeling and cardiac dysfunction. D + Q treatment efficiently eliminated senescent cells, rescuing CSC function, which resulted in functional myocardial repair/regeneration, improving cardiac function in murine DM. In conclusion, DM hampers CSC biology, inhibiting CSCs' regenerative potential through the induction of cellular senescence and SASP independently from aging. Senolytics clear senescence, abrogating the SASP and restoring a fully proliferative/differentiation-competent hCSC pool in T2DM with normalization of cardiac function.

In vitro CSC-derived cardiomyocytes exhibit the typical microRNA-mRNA blueprint of endogenous cardiomyocytes.

miRNAs modulate cardiomyocyte specification by targeting mRNAs of cell cycle regulators and acting in cardiac muscle lineage gene regulatory loops. It is unknown if or to-what-extent these miRNA/mRNA networks are operative during cardiomyocyte differentiation of adult cardiac stem/progenitor cells (CSCs). Clonally-derived mouse CSCs differentiated into contracting cardiomyocytes in vitro (iCMs). Comparison of "CSCs vs. iCMs" mRNome and microRNome showed a balanced up-regulation of CM-related mRNAs together with a down-regulation of cell cycle and DNA replication mRNAs. The down-regulation of cell cycle genes and the up-regulation of the mature myofilament genes in iCMs reached intermediate levels between those of fetal and neonatal cardiomyocytes. Cardiomyo-miRs were up-regulated in iCMs. The specific networks of miRNA/mRNAs operative in iCMs closely resembled those of adult CMs (aCMs). miR-1 and miR-499 enhanced myogenic commitment toward terminal differentiation of iCMs. In conclusions, CSC specification/differentiation into contracting iCMs follows known cardiomyo-MiR-dependent developmental cardiomyocyte differentiation trajectories and iCMs transcriptome/miRNome resembles that of CMs.

Rhythm dynamics of the aging heart: an experimental study using conscious, restrained mice.

Heart rate variability (HRV) is a measure of variation in time interval between heartbeats and reflects the influence of autonomic nervous system and circulating/locally released factors on sinoatrial node discharge. Here, we tested whether electrocardiograms (ECGs) obtained in conscious, restrained mice, a condition that affects sympathovagal balance, reveal alterations of heart rhythm dynamics with aging. Moreover, based on emergence of sodium channels as modulators of pacemaker activity, we addressed consequences of altered sodium channels on heart rhythm. C57Bl/6 mice and mice with enhanced late sodium current due to Nav1.5 mutation at Ser571 (S571E) at ~4 to ~24 mo of age, were studied. HRV was assessed using time- and frequency-domain and nonlinear parameters. For C57Bl/6 and S571E mice, standard deviation of RR intervals (SDRR), total power of RR interval variation, and nonlinear standard deviation 2 (SD2) were maximal at ~4 mo and decreased at ~18 and ~24 mo, together with attenuation of indexes of sympathovagal balance. Modulation of sympathetic and/or parasympathetic divisions revealed attenuation of autonomic tone at ~24 mo. At ~4 mo, S571E mice presented lower heart rate and higher SDRR, total power, and SD2 with respect to C57Bl/6, properties reversed by late sodium current inhibition. At ~24 mo, heart rate decreased in C57Bl/6 but increased in S571E, a condition preserved after autonomic blockade. Collectively, our data indicate that aging is associated with reduced HRV. Moreover, sodium channel function conditions heart rate and its age-related adaptations, but does not interfere with HRV decline occurring with age.NEW & NOTEWORTHY We have investigated age-associated alterations of heart rate properties in mice using conscious electrocardiographic recordings. Our findings support the notion that aging is coupled with altered sympathovagal balance with consequences on heart rate variability. Moreover, by using a genetically engineered mouse line, we provide evidence that sodium channels modulate heart rate and its age-related adaptations.

Targeting Cardiac Stem Cell Senescence to Treat Cardiac Aging and Disease.

Adult stem/progenitor are a small population of cells that reside in tissue-specific niches and possess the potential to differentiate in all cell types of the organ in which they operate. Adult stem cells are implicated with the homeostasis, regeneration, and aging of all tissues. Tissue-specific adult stem cell senescence has emerged as an attractive theory for the decline in mammalian tissue and organ function during aging. Cardiac aging, in particular, manifests as functional tissue degeneration that leads to heart failure. Adult cardiac stem/progenitor cell (CSC) senescence has been accordingly associated with physiological and pathological processes encompassing both non-age and age-related decline in cardiac tissue repair and organ dysfunction and disease. Senescence is a highly active and dynamic cell process with a first classical hallmark represented by its replicative limit, which is the establishment of a stable growth arrest over time that is mainly secondary to DNA damage and reactive oxygen species (ROS) accumulation elicited by different intrinsic stimuli (like metabolism), as well as external stimuli and age. Replicative senescence is mainly executed by telomere shortening, the activation of the p53/p16INK4/Rb molecular pathways, and chromatin remodeling. In addition, senescent cells produce and secrete a complex mixture of molecules, commonly known as the senescence-associated secretory phenotype (SASP), that regulate most of their non-cell-autonomous effects. In this review, we discuss the molecular and cellular mechanisms regulating different characteristics of the senescence phenotype and their consequences for adult CSCs in particular. Because senescent cells contribute to the outcome of a variety of cardiac diseases, including age-related and unrelated cardiac diseases like diabetic cardiomyopathy and anthracycline cardiotoxicity, therapies that target senescent cell clearance are actively being explored. Moreover, the further understanding of the reversibility of the senescence phenotype will help to develop novel rational therapeutic strategies.

Amelioration of diastolic dysfunction by dapagliflozin in a non-diabetic model involves coronary endothelium.

The results of trials with sodium-glucose cotransporter 2 (SGLT2) inhibitors raised the possibility that this class of drugs provides cardiovascular benefits independently from their anti-diabetic effects, although the mechanisms are unknown. Therefore, we tested the effects of SGLT2 inhibitor dapagliflozin on the progression of experimental heart disease in a non-diabetic model of heart failure with preserved ejection fraction. Dahl salt-sensitive rats were fed a high-salt diet to induce hypertension and diastolic dysfunction and were then treated with dapagliflozin for six weeks. Dapagliflozin ameliorated diastolic function as documented by echo-Doppler and heart catheterization, while blood pressure remained markedly elevated. Chronic in vivo treatment with dapagliflozin reduced diastolic Ca2+ and Na+ overload and increased Ca2+ transient amplitude in ventricular cardiomyocytes, although no direct action of dapagliflozin on isolated cardiomyocytes was observed. Dapagliflozin reversed endothelial activation and endothelial nitric oxide synthase deficit, with reduced cardiac inflammation and consequent attenuation of pro-fibrotic signaling. The potential involvement of coronary endothelium was supported by the endothelial upregulation of Na+/H+ exchanger 1in vivo and direct effects on dapagliflozin on the activity of this exchanger in endothelial cells in vitro. In conclusions, several mechanisms may cumulatively play a significant role in the dapagliflozin-associated cardioprotection. Dapagliflozin ameliorates diastolic function and exerts a positive effect on the myocardium, possibly targeting coronary endothelium. The lower degree of endothelial dysfunction, inflammation and fibrosis translate into improved myocardial performance.

Adult Cardiac Stem Cell Aging: A Reversible Stochastic Phenomenon?

Aging is by far the dominant risk factor for the development of cardiovascular diseases, whose prevalence dramatically increases with increasing age reaching epidemic proportions. In the elderly, pathologic cellular and molecular changes in cardiac tissue homeostasis and response to injury result in progressive deteriorations in the structure and function of the heart. Although the phenotypes of cardiac aging have been the subject of intense study, the recent discovery that cardiac homeostasis during mammalian lifespan is maintained and regulated by regenerative events associated with endogenous cardiac stem cell (CSC) activation has produced a crucial reconsideration of the biology of the adult and aged mammalian myocardium. The classical notion of the adult heart as a static organ, in terms of cell turnover and renewal, has now been replaced by a dynamic model in which cardiac cells continuously die and are then replaced by CSC progeny differentiation. However, CSCs are not immortal. They undergo cellular senescence characterized by increased ROS production and oxidative stress and loss of telomere/telomerase integrity in response to a variety of physiological and pathological demands with aging. Nevertheless, the old myocardium preserves an endogenous functionally competent CSC cohort which appears to be resistant to the senescent phenotype occurring with aging. The latter envisions the phenomenon of CSC ageing as a result of a stochastic and therefore reversible cell autonomous process. However, CSC aging could be a programmed cell cycle-dependent process, which affects all or most of the endogenous CSC population. The latter would infer that the loss of CSC regenerative capacity with aging is an inevitable phenomenon that cannot be rescued by stimulating their growth, which would only speed their progressive exhaustion. The resolution of these two biological views will be crucial to design and develop effective CSC-based interventions to counteract cardiac aging not only improving health span of the elderly but also extending lifespan by delaying cardiovascular disease-related deaths.

Novel Methods for High-resolution Assessment of Cardiac Action Potential Repolarization.

The profile of the action potential (AP) of cardiomyocytes contributes to the modality of ventricular repolarization of the heart. Experimentally, the examination of the AP in isolated cardiomyocytes provides information on their electrical properties, adaptations to physiological and pathological conditions, and putative ionic mechanisms involved in the process. Currently, there are no available platforms for automated assessment of AP properties and standard methodologies restrict the examination of the AP repolarization to discrete, user-defined ranges, neglecting significant intervals of the electrical recovery. This study proposes two automatic methods to assess AP profile throughout the entire repolarization phase. One method is based on AP data inversion and direct extraction of patterns describing beat-to-beat dynamics. The second method is based on evolutive singular value decomposition (ESVD), which identifies common patterns in a series of consecutive APs. The two methodologies were employed to analyze electrical signals collected from cardiomyocites obtained from healthy mice and animals with diabetes, a condition associated with alterations of AP properties in cardiac cells. Our methodologies revealed that the duration of the early repolarization phase of the AP tended to become progressively longer during a stimulation train, whereas the late repolarization progressively shortened. Although this behavior was comparable in the two groups of cells, alterations in AP dynamics occurred at distinct repolarization levels, a feature highlighted by the ESVD approach. In conclusion, the proposed methodologies allow detailed, automatic analysis of the AP repolarization and identification of critical alterations occurring in the electrical behavior of myocytes under pathological conditions.

Pressure Overload in Mice With Haploinsufficiency of Striated Preferentially Expressed Gene Leads to Decompensated Heart Failure.

Striated preferentially expressed gene (Speg) is a member of the myosin light chain kinase family of proteins. Constitutive Speg deficient (Speg-/-) mice develop a dilated cardiomyopathy, and the majority of these mice die in utero or shortly after birth. In the present study we assessed the importance of Speg in adult mice. Speg-/- mice that survived to adulthood, or adult striated muscle-specific Speg knockout mice (Speg-KO), demonstrated cardiac dysfunction and evidence of increased left ventricular (LV) internal diameter and heart to body weight ratio. To determine whether heterozygosity of Speg interferes with the response of the heart to pathophysiologic stress, Speg+/- mice were exposed to pressure overload induced by transverse aortic constriction (TAC). At baseline, Speg+/+ and Speg+/- hearts showed no difference in cardiac function. However, 4 weeks after TAC, Speg+/- mice had a marked reduction in LV function. This defect was associated with an increase in LV internal diameter and enhanced heart weight to body weight ratio, compared with Speg+/+ mice after TAC. The response of Speg+/- mice to pressure overload also included increased fibrotic deposition in the myocardium, disruption of transverse tubules, and attenuation in cell contractility, compared with Speg+/+ mice. Taken together, these data demonstrate that Speg is necessary for normal cardiac function and is involved in the complex adaptation of the heart in response to TAC. Haploinsufficiency of Speg results in decompensated heart failure when exposed to pressure overload.

Notch signaling modulates the electrical behavior of cardiomyocytes.

Notch receptor signaling is active during cardiac development and silenced in myocytes after birth. Conversely, outward K+ Kv currents progressively appear in postnatal myocytes leading to shortening of the action potential (AP) and acquisition of the mature electrical phenotype. In the present study, we tested the possibility that Notch signaling modulates the electrical behavior of cardiomyocytes by interfering with Kv currents. For this purpose, the effects of Notch receptor activity on electrophysiological properties of myocytes were evaluated using transgenic mice with inducible expression of the Notch1 intracellular domain (NICD), the functional fragment of the activated Notch receptor, and in neonatal myocytes after inhibition of the Notch transduction pathway. By patch clamp, NICD-overexpressing cells presented prolonged AP duration and reduced upstroke amplitude, properties that were coupled with reduced rapidly activating Kv and fast Na+ currents, compared with cells obtained from wild-type mice. In cultured neonatal myocytes, inhibition of the proteolitic release of NICD with a γ-secretase antagonist increased transcript levels of the Kv channel-interacting proteins 2 (KChIP2) and enhanced the density of Kv currents. Collectively, these results indicate that Notch signaling represents an important regulator of the electrophysiological behavior of developing and adult myocytes by repressing, at least in part, repolarizing Kv currents. NEW & NOTEWORTHY We investigated the effects of Notch receptor signaling on the electrical properties of cardiomyocytes. Our results indicate that the Notch transduction pathway interferes with outward K+ Kv currents, critical determinants of the electrical repolarization of myocytes.

Peptidyl-Prolyl Isomerase 1 Regulates Ca2+ Handling by Modulating Sarco(Endo)Plasmic Reticulum Calcium ATPase and Na2+/Ca2+ Exchanger 1 Protein Levels and Function.

BACKGROUND: Aberrant Ca2+ handling is a prominent feature of heart failure. Elucidation of the molecular mechanisms responsible for aberrant Ca2+ handling is essential for the development of strategies to blunt pathological changes in calcium dynamics. The peptidyl-prolyl cis-trans isomerase peptidyl-prolyl isomerase 1 (Pin1) is a critical mediator of myocardial hypertrophy development and cardiac progenitor cell cycle. However, the influence of Pin1 on calcium cycling regulation has not been explored. On the basis of these findings, the aim of this study is to define Pin1 as a novel modulator of Ca2+ handling, with implications for improving myocardial contractility and potential for ameliorating development of heart failure. METHODS AND RESULTS: Pin1 gene deletion or pharmacological inhibition delays cytosolic Ca2+ decay in isolated cardiomyocytes. Paradoxically, reduced Pin1 activity correlates with increased sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) and Na2+/Ca2+ exchanger 1 protein levels. However, SERCA2a ATPase activity and calcium reuptake were reduced in sarcoplasmic reticulum membranes isolated from Pin1-deficient hearts, suggesting that Pin1 influences SERCA2a function. SERCA2a and Na2+/Ca2+ exchanger 1 associated with Pin1, as revealed by proximity ligation assay in myocardial tissue sections, indicating that regulation of Ca2+ handling within cardiomyocytes is likely influenced through Pin1 interaction with SERCA2a and Na2+/Ca2+ exchanger 1 proteins. CONCLUSIONS: Pin1 serves as a modulator of SERCA2a and Na2+/Ca2+ exchanger 1 Ca2+ handling proteins, with loss of function resulting in impaired cardiomyocyte relaxation, setting the stage for subsequent investigations to assess Pin1 dysregulation and modulation in the progression of heart failure.

Phenotypically heterogeneous podoplanin-expressing cell populations are associated with the lymphatic vessel growth and fibrogenic responses in the acutely and chronically infarcted myocardium.

Cardiac lymphatic vasculature undergoes substantial expansion in response to myocardial infarction (MI). However, there is limited information on the cellular mechanisms mediating post-MI lymphangiogenesis and accompanying fibrosis in the infarcted adult heart. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFRα, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFRß or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. Thus, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development.

p53 Modulates the Fate of Cardiac Progenitor Cells Ex Vivo and in the Diabetic Heart In Vivo.

p53 is an important modulator of stem cell fate, but its role in cardiac progenitor cells (CPCs) is unknown. Here, we tested the effects of a single extra-copy of p53 on the function of CPCs in the presence of oxidative stress mediated by doxorubicin in vitro and type-1 diabetes in vivo. CPCs were obtained from super-p53 transgenic mice (p53-tg), in which the additional allele is regulated in a manner similar to the endogenous protein. Old CPCs with increased p53 dosage showed a superior ability to sustain oxidative stress, repair DNA damage and restore cell division. With doxorubicin, a larger fraction of CPCs carrying an extra-copy of the p53 allele recruited γH2A.X reestablishing DNA integrity. Enhanced p53 expression resulted in a superior tolerance to oxidative stress in vivo by providing CPCs with defense mechanisms necessary to survive in the milieu of the diabetic heart; they engrafted in regions of tissue injury and in three days acquired the cardiomyocyte phenotype. The biological advantage provided by the increased dosage of p53 in CPCs suggests that this genetic strategy may be translated to humans to increase cellular engraftment and growth, critical determinants of successful cell therapy for the failing heart.

Single-cell analysis of the fate of c-kit-positive bone marrow cells.

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

Hyperglycemia induces defective Ca2+ homeostasis in cardiomyocytes.

Diabetes and other metabolic conditions characterized by elevated blood glucose constitute important risk factors for cardiovascular disease. Hyperglycemia targets myocardial cells rendering ineffective mechanical properties of the heart, but cellular alterations dictating the progressive deterioration of cardiac function with metabolic disorders remain to be clarified. In the current study, we examined the effects of hyperglycemia on cardiac function and myocyte physiology by employing mice with high blood glucose induced by administration of streptozotocin, a compound toxic to insulin-producing ß-cells. We found that hyperglycemia initially delayed the electrical recovery of the heart, whereas cardiac function became defective only after ~2 mo with this condition and gradually worsened with time. Prolonged hyperglycemia was associated with increased chamber dilation, thinning of the left ventricle (LV), and myocyte loss. Cardiomyocytes from hyperglycemic mice exhibited defective Ca2+ transients before the appearance of LV systolic defects. Alterations in Ca2+ transients involved enhanced spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR), reduced cytoplasmic Ca2+ clearance, and declined SR Ca2+ load. These defects have important consequences on myocyte contraction, relaxation, and mechanisms of rate adaptation. Collectively, our data indicate that hyperglycemia alters intracellular Ca2+ homeostasis in cardiomyocytes, hindering contractile activity and contributing to the manifestation of the diabetic cardiomyopathy. NEW & NOTEWORTHY: We have investigated the effects of hyperglycemia on cardiomyocyte physiology and ventricular function. Our results indicate that defective Ca2+ handling is a critical component of the progressive deterioration of cardiac performance of the diabetic heart.

Reduction in Kv Current Enhances the Temporal Dispersion of the Action Potential in Diabetic Myocytes: Insights From a Novel Repolarization Algorithm.

BACKGROUND: Diabetes is associated with prolongation of the QT interval of the electrocardiogram and enhanced dispersion of ventricular repolarization, factors that, together with atherosclerosis and myocardial ischemia, may promote the occurrence of electrical disorders. Thus, we tested the possibility that alterations in transmembrane ionic currents reduce the repolarization reserve of myocytes, leading to action potential (AP) prolongation and enhanced beat-to-beat variability of repolarization. METHODS AND RESULTS: Diabetes was induced in mice with streptozotocin (STZ), and effects of hyperglycemia on electrical properties of whole heart and myocytes were studied with respect to an untreated control group (Ctrl) using electrocardiographic recordings in vivo, ex vivo perfused hearts, and single-cell patch-clamp analysis. Additionally, a newly developed algorithm was introduced to obtain detailed information of the impact of high glucose on AP profile. Compared to Ctrl, hyperglycemia in STZ-treated animals was coupled with prolongation of the QT interval, enhanced temporal dispersion of electrical recovery, and susceptibility to ventricular arrhythmias, defects observed, in part, in the Akita mutant mouse model of type I diabetes. AP was prolonged and beat-to-beat variability of repolarization was enhanced in diabetic myocytes, with respect to Ctrl cells. Density of Kv K(+) and L-type Ca(2+) currents were decreased in STZ myocytes, in comparison to cells from normoglycemic mice. Pharmacological reduction of Kv currents in Ctrl cells lengthened AP duration and increased temporal dispersion of repolarization, reiterating features identified in diabetic myocytes. CONCLUSIONS: Reductions in the repolarizing K(+) currents may contribute to electrical disturbances of the diabetic heart.

Myocyte repolarization modulates myocardial function in aging dogs.

Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions.