phosphorus-31-magnetic-resonance-spectroscopy-for-serial-measurements

Phosphorus-31 Magnetic Resonance Spectroscopy for Serial Measurements of Cardiac Energetics During Temperature-Controlled Heart Preservation

Reliable organ preservation is critical to increasing donor availability and improving transplant outcomes. While static storage remains the standard for ...

Johns Hopkins University School of Medicine

One key element of natural pacemakers is the pacemaker current encoded by the hyperpolarization-activated nucleotide-gated channel (HCN) gene family. Although HCN gene transfer has been used to engineer biological pacemakers, this strategy may be confounded by unpredictable consequences of heteromultimerization with endogenous HCN family members and limited flexibility with regard to frequency tuning of the engineered pacemaker.

Gene transfer of a synthetic pacemaker channel into the heart: a novel strategy for biological pacing.

Cardiac rhythm-associated disorders are caused by mal-functions of impulse generation and conduction. Present therapies for the impulse generation span a wide array of approaches but remain largely palliative. The progress in the understanding of the biology of the diseases with related biological tools beckons for new approaches to provide better alternatives to the present routine. Here, we review the current state of the art in gene- and cell-based approaches to correct cardiac rhythm disturbances. These include genetic suppression of an ionic current, stem cell therapies, adult somatic cell-fusion approach, novel synthetic pacemaker channel, and creating a self-contained pacemaker activity in non-excitable cells. We then conclude by discussing advantages and disadvantages of the new possibilities.

Creation of a biological pacemaker by gene- or cell-based approaches.

As an alternative to electronic pacemakers, we explored the feasibility of converting ventricular myocytes into pacemakers by somatic cell fusion. The idea is to create chemically induced fusion between myocytes and syngeneic fibroblasts engineered to express HCN1 pacemaker channels (HCN1-fibroblasts). HCN1-fibroblasts were fused with freshly isolated guinea pig ventricular myocytes using polyethylene-glycol 1500. In vivo fused myocyte-HCN1-fibroblast cells exhibited spontaneously oscillating action potentials; the firing frequency increased with beta-adrenergic stimulation. The heterokaryons created ectopic ventricular pacemaker activity in vivo at the site of cell injection. Coculture of nonfused HCN1-fibroblasts and myocytes without polyethylene-glycol 1500 revealed no evidence of dye transfer, demonstrating that the I(f)-mediated pacemaker activity arises from heterokaryons rather than electrotonic coupling. This nonviral, non-stem cell approach enables autologous, adult somatic cell therapy to create biopacemakers.

Creation of a biological pacemaker by cell fusion.

Calcium cycling figures prominently in excitation-contraction coupling and in various signaling cascades involved in the development of left ventricular hypertrophy. We hypothesized that genetic suppression of the L-type calcium channel accessory beta-subunit would modulate calcium current and suppress cardiac hypertrophy. A short hairpin RNA template sequence capable of mediating the knockdown of the L-type calcium channel accessory beta-subunit gene was incorporated into a lentiviral vector (PPT.CG.H1.beta(2)). Transduction of ventricular myocytes in vivo with the active short hairpin RNA partially inhibited the L-type calcium current. In neonatal rat cardiomyocytes, L-type calcium channel accessory beta-subunit gene knockdown reduced calcium transient amplitude. Similarly, [(3)H]leucine incorporation was attenuated in PPT.CG.H1.beta(2)-transduced neonatal rat cardiomyocytes compared with nonsilencing controls in a phenylephrine-induced hypertrophy model. In vivo gene transfer attenuated the hypertrophic response in an aortic-banded rat model of left ventricular hypertrophy, with reduced left ventricular wall thickness and heart weight/body weight ratios in PPT.CG.H1.beta(2)-injected rats at four weeks post transduction. Fractional shortening was preserved in rats treated with PPT.CG.H1.beta(2). These findings indicate that knockdown of L-type calcium channel accessory beta-subunit is capable of attenuating the hypertrophic response both in vitro and in vivo without compromising systolic performance. Suppression of the calcium channel beta subunit may represent a novel and useful therapeutic strategy for left ventricular hypertrophy.

Gene therapy to inhibit the calcium channel beta subunit: physiological consequences and pathophysiological effects in models of cardiac hypertrophy.

Cardiac rhythm disorders are caused by malfunctions of impulse generation or conduction. Malfunctions of impulse generation, that is, defects in pacemaking, are often life-threatening. Present therapies span a wide array of approaches, but remain largely palliative. Recent progress in understanding of the underlying biology of pacemaking opens up new prospects for better alternatives to the present routine. Specifically, development and use of biological pacemakers could prove to be advantageous to the conventional approaches.

Biological pacemakers as a therapy for cardiac arrhythmias.

Cardiac pacemaker cells of the sinoatrial node initiate each and every heartbeat. Compared with our understanding of the constituents of their electrical excitation, little is known about the metabolic underpinnings that drive the automaticity of pacemaker myocytes. This lack is largely owing to the scarcity of native cardiac pacemaker myocytes. Here, we take advantage of induced pacemaker myocytes generated by TBX18-mediated reprogramming (TBX18-iPMs) to investigate comparative differences in the metabolic program between pacemaker myocytes and working cardiomyocytes. TBX18-iPMs were more resistant to metabolic stresses, exhibiting higher cell viability upon oxidative stress. TBX18-induced pacemaker myocytes (iPMs) expensed a lower degree of oxidative phosphorylation and displayed a smaller capacity for glycolysis compared with control ventricular myocytes. Furthermore, the mitochondria were smaller in TBX18-iPMs than in the control. We reasoned that a shift in the balance between mitochondrial fusion and fission was responsible for the smaller mitochondria observed in TBX18-iPMs. We identified a mitochondrial inner membrane fusion protein, Opa1, as one of the key mediators of this process and demonstrated that the suppression of Opa1 expression increases the rate of synchronous automaticity in TBX18-iPMs. Taken together, our data demonstrate that TBX18-iPMs exhibit a low metabolic demand that matches their mitochondrial morphology and ability to withstand metabolic insult.

Induced cardiac pacemaker cells survive metabolic stress owing to their low metabolic demand.

Previously, we reported that heterologous expression of an embryonic transcription factor, Tbx18, reprograms ventricular cardiomyocytes into induced pacemaker cells (Tbx18-iPMs), though the key pathways are unknown. Here, we have used a tandem mass tag proteomic approach to characterize the impact of Tbx18 on neonatal rat ventricular myocytes. Tbx18 expression triggered vast proteome remodeling. Tbx18-iPMs exhibited increased expression of known pacemaker ion channels, including Hcn4 and Cx45 as well as upregulation of the mechanosensitive ion channels Piezo1, Trpp2 (PKD2), and TrpM7. Metabolic pathways were broadly downregulated, as were ion channels associated with ventricular excitation-contraction coupling. Tbx18-iPMs also exhibited extensive intracellular cytoskeletal and extracellular matrix remodeling, including 96 differentially expressed proteins associated with the epithelial-to-mesenchymal transition (EMT). RNAseq extended coverage of low abundance transcription factors, revealing upregulation of EMT-inducing Snai1, Snai2, Twist1, Twist2, and Zeb2. Finally, network diffusion mapping of &gt;200 transcriptional regulators indicates EMT and heart development factors occupy adjacent network neighborhoods downstream of Tbx18 but upstream of metabolic control factors. In conclusion, transdifferentiation of cardiac myocytes into pacemaker cells entails massive electrogenic, metabolic, and cytostructural remodeling. Structural changes exhibit hallmarks of the EMT. The results aid ongoing efforts to maximize the yield and phenotypic stability of engineered biological pacemakers.

Tbx18 Orchestrates Cytostructural Transdifferentiation of Cardiomyocytes to Pacemaker Cells by Recruiting the Epithelial-Mesenchymal Transition Program.

Complete atrioventricular block (CAVB) is a largely intractable disease that leads to severe bradyarrhythmia. The only treatment is the implantation of a pacemaker device. Left untreated, CAVB patients experience QT prolongation and ventricular overload, increasing susceptibility to cardiac remodeling and heart failure, as well as potentially lethal tachyarrhythmias. Animal models of CAVB offer a direct avenue to investigate the disease and potential disease-modifying therapies. However, existing models have limitations due to phenotype instability and high attrition rate. We aimed to create an improved method for disease model creation and report beating rate and variability analysis of the model. We report a modified surgical model of CAVB in rats by adapting a clinical radiofrequency energy generator to ablate the atrioventricular node region of the rat heart. Compared to previous ablation methods that utilize sharp needle entry into the AV node (AVN) region, the modified method resulted in a significantly higher success rate with a lower attrition rate. The rat model of CAVB showed stable conduction block for at least four weeks after model creation, supporting their suitability as a preclinical model of severe bradyarrhythmia due to AV conduction block. Models showed severe beat-to-beat variability with decreased overall autonomic innervation. We present a rodent model of a complete and stable AV block, which enhances the rigor and reproducibility of the disease model and downstream applications.

A highly effective, rodent model of complete heart block by epicardial radiofrequency ablation.

Cardiac arrhythmias increase during acute SARS-CoV-2 infection and in long COVID syndrome, by unknown mechanisms. This study explored the acute and long-term effects of COVID-19 on cardiac electrophysiology and the cardiac conduction system (CCS) in a hamster model. Electrocardiograms and subpleural pressures were recorded by telemetry for 4 weeks after SARS-CoV-2 infection, and interferon-stimulated gene expression and macrophage infiltration of the CCS were assessed at 4 days and 4 weeks postinfection. COVID-19 induced pronounced tachypnea and cardiac arrhythmias, including bradycardia and persistent atrioventricular block, though no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped, indicating persistent CCS injury. COVID-19 induced cardiac cytokine expression, connexin mislocalization, and CCS macrophage remodeling. Interestingly, sterile innate immune activation by direct cardiac injection of polyinosinic:polycytidylic acid (PIC) induced arrhythmias similar to those of COVID-19. PIC strongly induced cytokine secretion and interferon signaling in hearts, human induced pluripotent stem cell-derived cardiomyocytes, and engineered heart tissues, accompanied by alterations in excitation-contraction coupling. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by JAK/STAT inhibition or a mitochondrially targeted antioxidant, indicating that SARS-CoV-2 infection indirectly leads to arrhythmias by innate immune activation and redox stress, which could have implications for long COVID syndrome.

Article	Year
Gene transfer of a synthetic pacemaker channel into the heart: a novel strategy for biological pacing. Circulation\| PubMed ID: 17030681	2006
Creation of a biological pacemaker by gene- or cell-based approaches. Medical & biological engineering & computing\| PubMed ID: 17262203	2007
Creation of a biological pacemaker by cell fusion. Circulation research\| PubMed ID: 17395872	2007
Gene therapy to inhibit the calcium channel beta subunit: physiological consequences and pathophysiological effects in models of cardiac hypertrophy. Circulation research\| PubMed ID: 17556655	2007
Biological pacemakers as a therapy for cardiac arrhythmias. Current opinion in cardiology\| PubMed ID: 18281827	2008
Induced cardiac pacemaker cells survive metabolic stress owing to their low metabolic demand. Experimental & molecular medicine\| PubMed ID: 31519870	2019
Tbx18 Orchestrates Cytostructural Transdifferentiation of Cardiomyocytes to Pacemaker Cells by Recruiting the Epithelial-Mesenchymal Transition Program. Journal of proteome research\| PubMed ID: 36006872	2022
A highly effective, rodent model of complete heart block by epicardial radiofrequency ablation. Scientific reports\| PubMed ID: 41258478	2025
Innate immune activation and mitochondrial ROS induce acute and persistent cardiac conduction system dysfunction after COVID-19. JCI insight\| PubMed ID: 41424383	2025

Hee Cheol Cho

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