BACKGROUND: Direct reprogramming of fibroblasts into chemically induced cardiomyocyte-like cells (CiCMs) through small molecules presents a promising cell source for cardiac regeneration and therapeutic development. However, the contaminating non-cardiomyocytes, primarily unconverted fibroblasts, reduce the effectiveness of CiCMs in various applications. This study investigated a metabolic selection approach using lactate to enrich CiCMs by exploiting the unique metabolic capability of cardiomyocytes to utilize lactate as an alternative energy source. METHODS: Primary mouse embryonic fibroblasts (pMEFs) were reprogrammed into CiCMs and subjected to a glucosedepleted, lactate-supplemented medium for 4 days. Afterward, cell viability was analyzed, and cardiomyocyte efficiency was assessed through the expression of cardiac-specific markers. Additionally, electrophysiological function was evaluated by examining drug-induced responses. RESULTS: The lactate treatment led to a significant decrease in the viability of non-cardiomyocytes (pMEF-LAC), while CiCMs (CiCM-LAC) showed minimal cell death. Specifically, the expression of all cardiac-related markers was increased in CiCM-LAC. Metabolically purified CiCMs exhibited enhanced contractile force and increased contraction frequency compared to non-purified CiCMs, as well as an elevated responsiveness to drugs. CONCLUSION This study demonstrates that lactate-based metabolic selection is an effective and practical approach for enriching CiCMs, offering a cost-effective alternative to other purification methods. The application of this strategy could potentially broaden the accessibility and utility of reprogrammed cardiomyocytes in cardiac regeneration and therapeutic development.

Background and Objectives: SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins play crucial roles as neuronal postsynaptic scaffolds. Alongside neuropsychiatric symptoms, individuals with SHANK3 mutations often exhibit symptoms related to dysfunctions in other organs, including the heart. However, detailed insights into the cardiac functions of Shank3 remain limited. This study aimed to characterize the cardiac phenotypes of Shank3-overexpressing transgenic mice and explore the underlying mechanisms. Methods: Cardiac histological analysis, electrocardiogram and echocardiogram recordings were conducted on Shank3-overexpressing transgenic mice. Electrophysiological properties, including action potentials and L-type Ca2+ channel (LTCC) currents, were measured in isolated cardiomyocytes. Ca2+ homeostasis was assessed by analyzing cytosolic Ca2+transients and sarcoplasmic reticulum Ca2+ contents. Depolarization-induced cell shortening was examined in cardiomyocytes. Immunoprecipitation followed by mass spectrometrybased identification was employed to identify proteins in the cardiac Shank3 interactome.Western blot and immunocytochemical analyses were conducted to identify changes in protein expression in Shank3-overexpressing transgenic cardiomyocytes. Results: The hearts of Shank3-overexpressing transgenic mice displayed reduced weight and increased fibrosis. In vivo, sudden cardiac death, arrhythmia, and contractility impairments were identified. Shank3-overexpressing transgenic cardiomyocytes showed prolonged action potential duration and increased LTCC current density. Cytosolic Ca2+ transients were increased with prolonged decay time, while sarcoplasmic reticulum Ca2+ contents remained normal. Cell shortening was augmented in Shank3-overexpressing transgenic cardiomyocytes. The cardiac Shank3 interactome comprised 78 proteins with various functions. Troponin I levels were down-regulated in Shank3-overexpressing transgenic cardiomyocytes. Conclusions This study revealed cardiac dysfunction in Shank3-overexpressing transgenic mice, potentially attributed to changes in Ca2+ homeostasis and contraction, with a notable reduction in troponin I.

BACKGROUND: Direct reprogramming of fibroblasts into chemically induced cardiomyocyte-like cells (CiCMs) through small molecules presents a promising cell source for cardiac regeneration and therapeutic development. However, the contaminating non-cardiomyocytes, primarily unconverted fibroblasts, reduce the effectiveness of CiCMs in various applications. This study investigated a metabolic selection approach using lactate to enrich CiCMs by exploiting the unique metabolic capability of cardiomyocytes to utilize lactate as an alternative energy source. METHODS: Primary mouse embryonic fibroblasts (pMEFs) were reprogrammed into CiCMs and subjected to a glucosedepleted, lactate-supplemented medium for 4 days. Afterward, cell viability was analyzed, and cardiomyocyte efficiency was assessed through the expression of cardiac-specific markers. Additionally, electrophysiological function was evaluated by examining drug-induced responses. RESULTS: The lactate treatment led to a significant decrease in the viability of non-cardiomyocytes (pMEF-LAC), while CiCMs (CiCM-LAC) showed minimal cell death. Specifically, the expression of all cardiac-related markers was increased in CiCM-LAC. Metabolically purified CiCMs exhibited enhanced contractile force and increased contraction frequency compared to non-purified CiCMs, as well as an elevated responsiveness to drugs. CONCLUSION This study demonstrates that lactate-based metabolic selection is an effective and practical approach for enriching CiCMs, offering a cost-effective alternative to other purification methods. The application of this strategy could potentially broaden the accessibility and utility of reprogrammed cardiomyocytes in cardiac regeneration and therapeutic development.

BACKGROUND: Direct reprogramming of fibroblasts into chemically induced cardiomyocyte-like cells (CiCMs) through small molecules presents a promising cell source for cardiac regeneration and therapeutic development. However, the contaminating non-cardiomyocytes, primarily unconverted fibroblasts, reduce the effectiveness of CiCMs in various applications. This study investigated a metabolic selection approach using lactate to enrich CiCMs by exploiting the unique metabolic capability of cardiomyocytes to utilize lactate as an alternative energy source. METHODS: Primary mouse embryonic fibroblasts (pMEFs) were reprogrammed into CiCMs and subjected to a glucosedepleted, lactate-supplemented medium for 4 days. Afterward, cell viability was analyzed, and cardiomyocyte efficiency was assessed through the expression of cardiac-specific markers. Additionally, electrophysiological function was evaluated by examining drug-induced responses. RESULTS: The lactate treatment led to a significant decrease in the viability of non-cardiomyocytes (pMEF-LAC), while CiCMs (CiCM-LAC) showed minimal cell death. Specifically, the expression of all cardiac-related markers was increased in CiCM-LAC. Metabolically purified CiCMs exhibited enhanced contractile force and increased contraction frequency compared to non-purified CiCMs, as well as an elevated responsiveness to drugs. CONCLUSION This study demonstrates that lactate-based metabolic selection is an effective and practical approach for enriching CiCMs, offering a cost-effective alternative to other purification methods. The application of this strategy could potentially broaden the accessibility and utility of reprogrammed cardiomyocytes in cardiac regeneration and therapeutic development.

Background and Objectives: SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins play crucial roles as neuronal postsynaptic scaffolds. Alongside neuropsychiatric symptoms, individuals with SHANK3 mutations often exhibit symptoms related to dysfunctions in other organs, including the heart. However, detailed insights into the cardiac functions of Shank3 remain limited. This study aimed to characterize the cardiac phenotypes of Shank3-overexpressing transgenic mice and explore the underlying mechanisms. Methods: Cardiac histological analysis, electrocardiogram and echocardiogram recordings were conducted on Shank3-overexpressing transgenic mice. Electrophysiological properties, including action potentials and L-type Ca2+ channel (LTCC) currents, were measured in isolated cardiomyocytes. Ca2+ homeostasis was assessed by analyzing cytosolic Ca2+transients and sarcoplasmic reticulum Ca2+ contents. Depolarization-induced cell shortening was examined in cardiomyocytes. Immunoprecipitation followed by mass spectrometrybased identification was employed to identify proteins in the cardiac Shank3 interactome.Western blot and immunocytochemical analyses were conducted to identify changes in protein expression in Shank3-overexpressing transgenic cardiomyocytes. Results: The hearts of Shank3-overexpressing transgenic mice displayed reduced weight and increased fibrosis. In vivo, sudden cardiac death, arrhythmia, and contractility impairments were identified. Shank3-overexpressing transgenic cardiomyocytes showed prolonged action potential duration and increased LTCC current density. Cytosolic Ca2+ transients were increased with prolonged decay time, while sarcoplasmic reticulum Ca2+ contents remained normal. Cell shortening was augmented in Shank3-overexpressing transgenic cardiomyocytes. The cardiac Shank3 interactome comprised 78 proteins with various functions. Troponin I levels were down-regulated in Shank3-overexpressing transgenic cardiomyocytes. Conclusions This study revealed cardiac dysfunction in Shank3-overexpressing transgenic mice, potentially attributed to changes in Ca2+ homeostasis and contraction, with a notable reduction in troponin I.

Background and Objectives: SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins play crucial roles as neuronal postsynaptic scaffolds. Alongside neuropsychiatric symptoms, individuals with SHANK3 mutations often exhibit symptoms related to dysfunctions in other organs, including the heart. However, detailed insights into the cardiac functions of Shank3 remain limited. This study aimed to characterize the cardiac phenotypes of Shank3-overexpressing transgenic mice and explore the underlying mechanisms. Methods: Cardiac histological analysis, electrocardiogram and echocardiogram recordings were conducted on Shank3-overexpressing transgenic mice. Electrophysiological properties, including action potentials and L-type Ca2+ channel (LTCC) currents, were measured in isolated cardiomyocytes. Ca2+ homeostasis was assessed by analyzing cytosolic Ca2+transients and sarcoplasmic reticulum Ca2+ contents. Depolarization-induced cell shortening was examined in cardiomyocytes. Immunoprecipitation followed by mass spectrometrybased identification was employed to identify proteins in the cardiac Shank3 interactome.Western blot and immunocytochemical analyses were conducted to identify changes in protein expression in Shank3-overexpressing transgenic cardiomyocytes. Results: The hearts of Shank3-overexpressing transgenic mice displayed reduced weight and increased fibrosis. In vivo, sudden cardiac death, arrhythmia, and contractility impairments were identified. Shank3-overexpressing transgenic cardiomyocytes showed prolonged action potential duration and increased LTCC current density. Cytosolic Ca2+ transients were increased with prolonged decay time, while sarcoplasmic reticulum Ca2+ contents remained normal. Cell shortening was augmented in Shank3-overexpressing transgenic cardiomyocytes. The cardiac Shank3 interactome comprised 78 proteins with various functions. Troponin I levels were down-regulated in Shank3-overexpressing transgenic cardiomyocytes. Conclusions This study revealed cardiac dysfunction in Shank3-overexpressing transgenic mice, potentially attributed to changes in Ca2+ homeostasis and contraction, with a notable reduction in troponin I.

BACKGROUND: Direct reprogramming of fibroblasts into chemically induced cardiomyocyte-like cells (CiCMs) through small molecules presents a promising cell source for cardiac regeneration and therapeutic development. However, the contaminating non-cardiomyocytes, primarily unconverted fibroblasts, reduce the effectiveness of CiCMs in various applications. This study investigated a metabolic selection approach using lactate to enrich CiCMs by exploiting the unique metabolic capability of cardiomyocytes to utilize lactate as an alternative energy source. METHODS: Primary mouse embryonic fibroblasts (pMEFs) were reprogrammed into CiCMs and subjected to a glucosedepleted, lactate-supplemented medium for 4 days. Afterward, cell viability was analyzed, and cardiomyocyte efficiency was assessed through the expression of cardiac-specific markers. Additionally, electrophysiological function was evaluated by examining drug-induced responses. RESULTS: The lactate treatment led to a significant decrease in the viability of non-cardiomyocytes (pMEF-LAC), while CiCMs (CiCM-LAC) showed minimal cell death. Specifically, the expression of all cardiac-related markers was increased in CiCM-LAC. Metabolically purified CiCMs exhibited enhanced contractile force and increased contraction frequency compared to non-purified CiCMs, as well as an elevated responsiveness to drugs. CONCLUSION This study demonstrates that lactate-based metabolic selection is an effective and practical approach for enriching CiCMs, offering a cost-effective alternative to other purification methods. The application of this strategy could potentially broaden the accessibility and utility of reprogrammed cardiomyocytes in cardiac regeneration and therapeutic development.

Background and Objectives: SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins play crucial roles as neuronal postsynaptic scaffolds. Alongside neuropsychiatric symptoms, individuals with SHANK3 mutations often exhibit symptoms related to dysfunctions in other organs, including the heart. However, detailed insights into the cardiac functions of Shank3 remain limited. This study aimed to characterize the cardiac phenotypes of Shank3-overexpressing transgenic mice and explore the underlying mechanisms. Methods: Cardiac histological analysis, electrocardiogram and echocardiogram recordings were conducted on Shank3-overexpressing transgenic mice. Electrophysiological properties, including action potentials and L-type Ca2+ channel (LTCC) currents, were measured in isolated cardiomyocytes. Ca2+ homeostasis was assessed by analyzing cytosolic Ca2+transients and sarcoplasmic reticulum Ca2+ contents. Depolarization-induced cell shortening was examined in cardiomyocytes. Immunoprecipitation followed by mass spectrometrybased identification was employed to identify proteins in the cardiac Shank3 interactome.Western blot and immunocytochemical analyses were conducted to identify changes in protein expression in Shank3-overexpressing transgenic cardiomyocytes. Results: The hearts of Shank3-overexpressing transgenic mice displayed reduced weight and increased fibrosis. In vivo, sudden cardiac death, arrhythmia, and contractility impairments were identified. Shank3-overexpressing transgenic cardiomyocytes showed prolonged action potential duration and increased LTCC current density. Cytosolic Ca2+ transients were increased with prolonged decay time, while sarcoplasmic reticulum Ca2+ contents remained normal. Cell shortening was augmented in Shank3-overexpressing transgenic cardiomyocytes. The cardiac Shank3 interactome comprised 78 proteins with various functions. Troponin I levels were down-regulated in Shank3-overexpressing transgenic cardiomyocytes. Conclusions This study revealed cardiac dysfunction in Shank3-overexpressing transgenic mice, potentially attributed to changes in Ca2+ homeostasis and contraction, with a notable reduction in troponin I.

BACKGROUND: Direct reprogramming of fibroblasts into chemically induced cardiomyocyte-like cells (CiCMs) through small molecules presents a promising cell source for cardiac regeneration and therapeutic development. However, the contaminating non-cardiomyocytes, primarily unconverted fibroblasts, reduce the effectiveness of CiCMs in various applications. This study investigated a metabolic selection approach using lactate to enrich CiCMs by exploiting the unique metabolic capability of cardiomyocytes to utilize lactate as an alternative energy source. METHODS: Primary mouse embryonic fibroblasts (pMEFs) were reprogrammed into CiCMs and subjected to a glucosedepleted, lactate-supplemented medium for 4 days. Afterward, cell viability was analyzed, and cardiomyocyte efficiency was assessed through the expression of cardiac-specific markers. Additionally, electrophysiological function was evaluated by examining drug-induced responses. RESULTS: The lactate treatment led to a significant decrease in the viability of non-cardiomyocytes (pMEF-LAC), while CiCMs (CiCM-LAC) showed minimal cell death. Specifically, the expression of all cardiac-related markers was increased in CiCM-LAC. Metabolically purified CiCMs exhibited enhanced contractile force and increased contraction frequency compared to non-purified CiCMs, as well as an elevated responsiveness to drugs. CONCLUSION This study demonstrates that lactate-based metabolic selection is an effective and practical approach for enriching CiCMs, offering a cost-effective alternative to other purification methods. The application of this strategy could potentially broaden the accessibility and utility of reprogrammed cardiomyocytes in cardiac regeneration and therapeutic development.

The development of selective targeting of drug molecules towards the mitochondria is an important issue related to therapy efficacy. In this study, we report that gallic acid (GA)-mitochondria targeting sequence (MTS)-H3R9 exhibits a dual role as a mitochondria-targeting vehicle with antioxidant activity for disease therapy.In viability assays, GA-MTS-H3R9 showed a better rescue action compared to that of MTS-H3R9 . GA-MTS-H3R9 dramatically exhibited cell penetration and intercellular uptake compared to MTS and fit escape from lysosome release to the cytosol. We demonstrated the useful targeting of GA-MTS-H3R9 towards mitochondria in AC16 cells.Also, we observed that the antioxidant properties of mitochondrial-accrued GA-MTSH3R9 alleviated cell damage by reactive oxygen species production and disrupted mitochondrial membrane potential. GA-MTS-H3R9 showed a very increased cytoprotective effect against anticancer activity compared to that of MTS-H3R9 . We showed that GA-MTS-H3R9 can act as a vehicle for mitochondria-targeting and as a reagent for therapeutic applications intended for cardiovascular disease treatment.