Citations

Modulating immune function is a critical strategy in cancer and atherosclerosis treatments. For cancer, boosting or maintaining the immune system is crucial to prevent tumor growth. However, in vascular disease, mitigating immune responses can decrease inflammation and slow atherosclerosis progression. Anti-inflammatory therapy, therefore, presents a unique dilemma for cancer survivors: while it may decrease cardiovascular risk, it might also promote cancer growth and metastasis by suppressing the immune response. Senescence presents a potentially targetable solution to this challenge; senescence increases the risk of both cancer therapy resistance and vascular disease. Exercise, notably, shows promise in delaying this premature senescence, potentially improving cancer outcomes and lowering vascular disease risk post-treatment. This review focuses on the long-term impact of cancer therapies on vascular health. We underscore the importance of modulating senescence to balance cancer treatment’s effectiveness and its vascular impact, and we emphasize investigating the role of exercise-mediated suppression of senescence in improving cancer survivorship.

Background: Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young people. Recently, a patient with HCM with paroxysmal atrial fibrillation (AF) and sinoatrial block was found to have a missense mutation, E169K, in the junctophilin 2 (JPH2) protein. Loss of JPH2 has been shown to affect intracellular Ca2+ release in the heart. To date, no study has evaluated the effect of E169K mutation on Ca2+ handling and arrhythmogenesis. Hypothesis: The JPH2 E169K mutation promotes sarcoplasmic reticulum Ca2+ leak causing abnormal atrial conduction and AF. Methods and Results: We evaluated our hypothesis by generating 3 mice that overexpressed 1) JPH2 with E169K mutation (EK-Tg), 2) WT JPH2 (WT-Tg) and 3) JPH2 with inactive P584S mutation (PS-Tg). On Western Blot, JPH2 expression was increased by 2-3 fold in all the mouse groups compared to non-transgenic controls. Echocardiography at 6 months of age revealed normal fractional shortening in all mice. At baseline, EK-Tg mice had a shorter RR interval (104±1 ms, n=10) vs. WT-Tg (123±5 ms, n=8, p=0.001) and PS-Tg mice (121±5 ms, n=5, p<0.001). EK-Tg mice also had a shorter PR interval (36±1 ms, n=10) vs. WT-Tg (41±1 ms, n=8, p=0.01) and PS-Tg mice (39±1 ms, n=5, p=0.15). On intracardiac electrophysiology studies, EK-Tg mice had shorter AVERP (39±2 ms, n=10) vs. WT-Tg (51±2 ms, n=8, p=0.005) and PS-Tg mice (48±3 ms, n=5, p=0.05). On overdrive pacing of the atria in the presence of epinephrine and caffeine, EK-Tg mice had a 57% incidence of AF (n=10) vs. 21% in WT-Tg (n=8, p<0.05) and 33% in PS-Tg mice (n=5, p=0.2). Isolated cardiomyocytes from EK-Tg mice had a higher Ca2+ spark frequency (6.7±0.8 events/100 um/s, n=20) vs. cardiomyocytes from WT-Tg (1.8±0.5 events/100 um/s, n=10, p<0.001) and PS-Tg mice (3.3±1.0 events/100 um/s, n=16, p<0.05). Lastly, JPH2 levels were lower in RyR2 channels immunoprecipitated from EK-Tg mice vs. RyR2 from WT-Tg mice (65% vs. 100%, p<0.05). Conclusion: The JPH2 mutation, E169K, leads to reduced binding of JPH2 with RyR2, increased SR Ca2+ leak, abnormal atrial conduction and AF. The data suggest that JPH2 may serve as a critical stabilizing molecule for RyR2; further approaches to stabilize this interaction may lead to improved control of AF in susceptible populations.

Background. Na + /Ca 2+ exchanger (NCX) is essential in tuning intracellular Ca 2+ and the development of heart failure (HF). We reported that loss of Junctophilin 2 (JPH2) in adult mouse hearts leads to reduced NCX activity in addition to hyperactive RyR2s and more arrhythmogenic Ca 2+ waves. NCX has been found to co-localize with RyR2 recently. However, what is the physiological significance of junctional NCX and whether the reduced junctional NCX activity following JPH2 knockdown contributes to the arrhythmogenesis is still unknown. Hypothesis. Dysfunction of Junctional NCX in response to JPH2 knockdown alters spatial properties of Ca 2+ sparks and contributes to arrhythmogenesis. Methods. Tamoxifen-treated MCM and MCM-shJPH2 (KD) mice were studied. Contractile function was measured by echocardiography. Open probability of single RyR2 was evaluated by lipid bilayer. Ca 2+ spark were assessed with confocal microscopy. NCX activity was measured by exponential fitting of the declining phase of caffeine-induced Ca 2+ transient. Results. JPH2 knockdown increased spark size (MCM: 2.55 ± 0.14; KD: 4.25 ± 0.16, P < 0.001), suggesting an increased occurrence of proarrhythmic macrosparks. Cardiac Contractile function (EF% ) is negatively correlated with Ca 2+ spark size (n = 8, P < 0.05). Increased Open probability of RyR2 (MCM: 0.038 ± 0.018 KD: 0.274 ± 0.142, P < 0.01) is unlikely the reason for the ~70% increase in spark size, since it is not observed in 3 other mouse models with hyperactive RyR2s but normal JPH2 levels (RyR2:S2814D, mdx and FKBP12.6 -/- ). Loss of JPH2 also slows Ca 2+ efflux via NCX by ~50% (MCM: 2.70 ± 0.23 vs. KD: 5.11 ± 0.61 s, P < 0.01) without altering NCX expression level. Spark size and NCX activity are negatively correlated (n = 8 mice, P < 0.05), and partial inhibition (~30%) of NCX with 1 mM Cd 2+ significantly increased spark size by 14.0 ± 2.8% (n = 8, P <0.05) in wt mice. Conclusion. For the first time we showed that reduced NCX activity increases spatial properties of Ca 2+ sparks and thus arrhythmogenesis. Since reduced JPH2 is associated with HF and NCX plays more important role in human than in murine, reduced junctional NCX activity following loss of JPH2 could be a novel mechanism underlying the development of HF and associated ventricular arrhythmias.

AbstractBackgroundWolff–Parkinson–White (WPW) syndrome is a relatively common arrhythmia affecting ~1–3/1,000 individuals. Mutations in PRKAG2 have been described in rare patients in association with cardiomyopathy. However, the genetic basis of WPW in individuals with a structurally normal heart remains poorly understood. Sudden death due to atrial fibrillation (AF) can also occur in these individuals. Several studies have indicated that despite ablation of an accessory pathway, the risk of AF remains high in patients compared to general population.MethodsWe applied exome sequencing in 305 subjects, including 65 trios, 80 singletons, and 6 multiple affected families. We used de novo analysis, candidate gene approach, and burden testing to explore the genetic contributions to WPW.ResultsA heterozygous deleterious variant in PRKAG2 was identified in one subject, accounting for 0.6% (1/151) of the genetic basis of WPW in this study. Another individual with WPW and left ventricular hypertrophy carried a known pathogenic variant in MYH7. We found rare de novo variants in genes associated with arrhythmia and cardiomyopathy (ANK2, NEBL, PITX2, and PRDM16) in this cohort. There was an increased burden of rare deleterious variants (MAF ≤ 0.005) with CADD score ≥ 25 in genes linked to AF in cases compared to controls (P = .0023).ConclusionsOur findings show an increased burden of rare deleterious variants in genes linked to AF in WPW syndrome, suggesting that genetic factors that determine the development of accessory pathways may be linked to an increased susceptibility of atrial muscle to AF in a subset of patients.

Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca2+ leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca2+ release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca2+ imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca2+ leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na+/Ca2+-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd2+ administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca2+ leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca2+. This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca2+ leak and heart failure development.

Background— Cardiac dysfunction in failing hearts of human patients and animal models is associated with both microtubule densification and transverse-tubule (T-tubule) remodeling. Our objective was to investigate whether microtubule densification contributes to T-tubule remodeling and excitation–contraction coupling dysfunction in heart disease. Methods and Results— In a mouse model of pressure overload–induced cardiomyopathy by transaortic banding, colchicine, a microtubule depolymerizer, significantly ameliorated T-tubule remodeling and cardiac dysfunction. In cultured cardiomyocytes, microtubule depolymerization with nocodazole or colchicine profoundly attenuated T-tubule impairment, whereas microtubule polymerization/stabilization with taxol accelerated T-tubule remodeling. In situ immunofluorescence of heart tissue sections demonstrated significant disorganization of junctophilin-2 (JP2), a protein that bridges the T-tubule and sarcoplasmic reticulum membranes, in transaortic banded hearts as well as in human failing hearts, whereas colchicine injection significantly preserved the distribution of JP2 in transaortic banded hearts. In isolated mouse cardiomyocytes, prolonged culture or treatment with taxol resulted in pronounced redistribution of JP2 from T-tubules to the peripheral plasma membrane, without changing total JP2 expression. Nocodazole treatment antagonized JP2 redistribution. Moreover, overexpression of a dominant-negative mutant of kinesin 1, a microtubule motor protein responsible for anterograde trafficking of proteins, protected against JP2 redistribution and T-tubule remodeling in culture. Finally, nocodazole treatment improved Ca 2+ handling in cultured myocytes by increasing the amplitude of Ca 2+ transients and reducing the frequency of Ca 2+ sparks. Conclusion— Our data identify a mechanistic link between microtubule densification and T-tubule remodeling and reveal microtubule-mediated JP2 redistribution as a novel mechanism for T-tubule disruption, loss of excitation–contraction coupling, and heart failure.

Background— Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca 2+ channels and Ca 2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca 2+ -induced Ca 2+ release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. Methods and Results— To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma–sarcoplasmic reticulum distance. Conclusions— Loss of JPH2 had profound effects on Ca 2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca 2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA–mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca 2+ release in the heart.

Background— Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca 2+ ) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca 2+ channels and sarcoplasmic reticular ryanodine receptors. However, it remains unclear whether JPH2 expression is altered in patients with hypertrophic cardiomyopathy (HCM). In addition, the effects of downregulation of JPH2 expression on intracellular Ca 2+ handling are presently poorly understood. We sought to determine whether loss of JPH2 expression is noted among patients with HCM and whether expression silencing might perturb Ca 2+ handling in a prohypertrophic manner. Methods and Results— JPH2 expression was reduced in flash-frozen human cardiac tissue procured from patients with HCM compared with ostensibly healthy traumatic death victims. Partial silencing of JPH2 expression in HL-1 cells by a small interfering RNA probe targeted to murine JPH2 mRNA (shJPH2) resulted in myocyte hypertrophy and increased expression of known markers of cardiac hypertrophy. Whereas expression levels of major Ca 2+ -handling proteins were unchanged, shJPH2 cells demonstrated depressed maximal Ca 2+ transient amplitudes that were insensitive to L-type Ca 2+ channel activation with JPH2 knockdown. Further, reduced caffeine-triggered sarcoplasmic reticulum store Ca 2+ levels were observed with potentially increased total Ca 2+ stores. Spontaneous Ca 2+ oscillations were elicited at a higher extracellular [Ca 2+ ] and with decreased frequency in JPH2 knockdown cells. Conclusions— Our results show that JPH2 levels are reduced in patients with HCM. Reduced JPH2 expression results in reduced excitation-contraction coupling gain as well as altered Ca 2+ homeostasis, which may be associated with prohypertrophic remodeling.

Junctophilins (JPHs) are members of a junctional membrane complex protein family important for the physical approximation of plasmalemmal and sarcoplasmic/endoplasmic reticulum membranes. As such, JPHs facilitate signal transduction in excitable cells between plasmalemmal voltage-gated calcium channels and intracellular calcium release channels. To determine the molecular evolution of the JPH gene family, we performed a phylogenetic analysis of over 60 JPH genes from over 40 species and compared conservation across species and different isoforms. We found that JPHs are evolutionary highly conserved, in particular the membrane occupation and recognition nexus motifs found in all species. Our data suggest that an ancestral form of JPH arose at the latest in a common metazoan ancestor and that in vertebrates four isoforms arose, probably following two rounds of whole genome duplications. By combining multiple prediction techniques with sequence alignments, we also postulate the presence of new important functional regions and candidate sites for posttranslational modifications. The increasing number of available sequences yields significant insight into the molecular evolution of JPHs. Our analysis is consistent with the emerging concept that JPHs serve dual important functions in excitable cells: structural assembly of junctional membrane complexes and regulation of intracellular calcium signaling pathways.